Figure 1.
Family pedigrees and corresponding KISS1R mutations.
Panel A. Pedigree of the family with homozygous KISS1R c.937T>C mutation. The proband, subject II-8 (arrow), and his two affected sisters, subjects II-1 and II-3, were homozygous for the c.937T>C mutation. The unaffected mother (I-1) and father (I-2) were heterozygous for the same mutation. Two unaffected sisters, II-6 and II-7, carried two wild-type alleles. This missense mutation (p.Tyr313His, (H)) is located in the seventh transmembrane segment. Squares represent males and circles females. Solid symbols indicate affected subjects and half-shaded symbols unaffected heterozygous. Panel B. Pedigree of the family with compound heterozygous KISS1R mutations c.305T>C and c.1195T>A. The proband, subject II-1 (arrow), was a compound heterozygote for the KISS1R mutations c.305T>C and c.1195T>A. The unaffected mother (I-1) was heterozygous for the c.305T>C mutation. The unaffected father was deceased and therefore unavailable for genetic analysis. The c.305T>C substitution produces a missense mutation (p.Leu102Pro, (P)) in the second transmembrane segment of KISS1R. The c.1195T>A substitution abolishes the natural stop codon (p.Stop399Arg, (R)). Small black circles indicate miscarriages. Panel C. Results of automatic DNA KISS1R sequencing encompassing the c.937T>C homozygous mutation in the propositus, compared to an unaffected control (lower panel). Inframe amino acids are indicated above each sequence. Panel D. Schematic representation of KISS1R and location of the mutations. Panel E. Results of automatic DNA KISS1R sequencing encompassing the c.305T>C and c.1195T>A compound heterozygous mutations in the propositus. Inframe amino acids are indicated above each sequence.
Table 1.
Clinical and hormonal characteristics in affected patients with KISS1R mutations of two kindred.
Figure 2.
Pattern of LH secretion in a man with complete normosmic CHH due to KISS1R mutations (subject II-1, family 2).
In this individual with very low and nonpulsatile basal LH levels, LH pulsatility was restored by pulsatile GnRH administration. LH secretion was evaluated for 4 hours before GnRH treatment and also on day 16 of pulsatile GnRH administration. Arrows indicate the GnRH boluses and asterisks denote detectable pulses, as analyzed with Thomas' algorithm [7], [27]. Plasma testosterone (T) levels, which were very low before GnRH treatment, increased after pulsatile GnRH administration in this man, indicating testicular stimulation. Conversion to SI units: testosterone, nanograms per milliliter (x 3.467 = nanomoles per liter). The testosterone normal range in men is 2.8–9.0 ng/ml. IB: low serum inhibin B also increased after pulsatile GnRH administration.
Figure 3.
Modeling and functional consequences of the p.Tyr313His KISS1R mutation. Panel A.
Evolutionary conservation of Tyr313. Tyr313 is totally conserved among KISS1R homologs. Hs: Homo sapiens; Nl: Nomascus leucogenys; Cf: Canis familiaris; Bt: Bos taurus; Mm: Mus musculus; Rn: Rattus norvegicus; Sh: Sarcophilus harrisii; Oa: Ornithorhynchus anatinus; Xt: Xenopus tropicalis; Dr: Danio rerio; Tr: Takifugu rubripes. The substitution is indicated below. Panel B. Modeling of the transmembrane region of KISS1R. Tyrosine 313 (and its substitution by a histidine) are located at the bottom of the putative binding pocket for kisspeptin. Tyr313 is in a suitable position to form two hydrogen bonds with Cys95 and Thr99 (see upper insert), both located in the second transmembrane segment. The His substitution abolishes these interactions (lower insert). Panel C. Variation of intracytoplasmic calcium concentrations in WT, Tyr313His (Y313H) mutated KISS1R and empty vector. Transfected cells were stimulated by kisspeptin (10−7 M and 10−9 M): we observe a very significant decrease in calcium release in mutant-KISS1R transfected cells (***p<0.0001). Each point represents the area under the curve (AUC) for an individual cell. WT: black circles, Y313H: red squares, empty vector green circles. Panel D. ERK1/2 phosphorylation in WT and Y313H HEK-293 transfected cells. The upper panel shows the marked increase in ERK1/2 phosphorylation in cells transfected with the wild-type KISS1R, after 5 minutes of Kp-10 stimulation. The lower panel shows the absence in ERK1/2 phosphorylation when cells were transfected with the vector containing Y313H-mutated KISS1R. C: positive control: WT KISS1R stimulated by kisspeptin after 5 minutes. Representative figure of 3 independent experiments. Panel E. Kp-10 dose-response of the luc2P/SRE reporter. Increasing concentrations of Kp-10 led to a gradual increase in the luciferase activity of wild-type KISS1R (black circles). In contrast, the mutant KISS1R (red squares), like the empty vector (green circles), did not enhance luciferase activity.