TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction

The ethics committee of the Cukurova University Faculty of Medicine approved this study, and full informed consent was obtained for each participant. nIHH was defined by conventional criteria ( supplementary methods ). Normal karyotypes were confirmed and mutations in GNRHR , GNRH1 , GPR54 , KISS1 , KAL1 , NELF , PROK2 , PROK2R and FGFR1 genes were ruled out by sequencing. Whole genome SNP analysis used 250K NspI Affymetrix SNP microarrays (Affymetrix, CA, USA) and data were analyzed using AutoSNPa software 9 . PCR-amplified exons and splice junctions of TAC3 or TACR3 were sequenced on an ABI PRISM 3130 autosequencer (Applied Biosystems, Foster City, CA). For primer sequences and annealing temperatures see supplementary Table 2 . Wild type or mutant TACR3 in pIRES2-AcGFP1 (Clontech) were transfected into HEK293A cells (QBiogene) using polyethylenimine (Sigma) before seeding cells into poly-L-lysine-coated glass-bottomed dishes 16 hours later. At 24 hours cells were loaded with Fura2-AM for 30 min. Experiments were performed on an inverted fluorescence microscope (Eclipse TE2000, Nikon, UK) with a 40x oil-immersion objective. Fura2 was excited at 340, 360 and 380 nm, and GFP at 475 nm, using a 75W xenon arc lamp and a monochromator (Cairn Research, Faversham, UK) and MetaFluor software (Molecular Devices, UK). Emission, filtered at 510/80 and 535/25 for Fura2 and GFP respectively, was recorded with a QuantEM CCD camera (Photometrics, Roper Scientific, UK). NKB (Sigma) or custom made mutant NKB with or without C terminal amidation (New England Peptides) were perfused in bath solution at ~1 ml/min with a chamber volume of ~0.2 ml. 340/380 nm ratio was calculated from images taken at 100 ms excitation at each wavelength at 0.5 Hz. Free Ca 2+ -concentrations were calculated for individual cells after background subtraction using the equation of Grynkiewicz et al (1985) assuming a K D of 224 nM. Minimal and maximal signals were recorded in the presence of 5 mM ionomycin in 5 mM EGTA/0 mM Ca 2+ and 5 mM Ca 2+ respectively at the end of the experiment. Further detail is given in the supplementary methods .