Figure 1.
Family pedigrees and consequences of TACR3 mutations on NK3R structure.
Panel A. Pedigree of the family with homozygous TACR3 c.483_499 deletion. The proband, subject II-6 (arrow), and her two affected sisters (subjects II-5 and II-8), were homozygous for the c.483_499 deletion. The unaffected father (I-1) was heterozygous for the mutation. The unaffected sister (II-1) carried homozygous wild-type alleles. This deletion results in the emergence of a premature stop codon (Q161HfsX23), truncating NK3R after the third transmembrane segment. Panel B. Pedigree of the family with compound heterozygous TACR3 mutations c.824G>A and c.1003C>T. The proband, subject II-1 (arrow), was compound heterozygous for TACR3 mutations c.824G>A and c.1003C>T. The unaffected father (I-1) was heterozygous for the c.824G>A mutation and the unaffected mother was heterozygous for the c.1003C>T mutation. The c.824G>A substitution produces a stop codon in the 5th transmembrane segment (p.W275X) of NK3R. The c.1003C>T substitution produces a stop codon in the junction between the third extracellular loop and the seventh transmembrane domain (p.Q335X) of NK3R. Panel C. Pedigree of the family with compound heterozygous TACR3 mutations c.799T>A and c.824G>A. The proband, subject II-1 (arrow), was compound heterozygous for TACR3 mutations c.799T>A and c.824G>A. The unaffected father (I-1) was heterozygous for the recurent c.824G>A mutation and the unaffected mother was heterozygous for the c.799T>A mutation. This latter mutation affects a conserved amino acid in the fifth transmembrane domain (p.Y267N). Solid symbols indicate affected subjects and half-shaded symbols indicate unaffected heterozygotes. Circles represent female family members and squares male family members.
Table 1.
One TAC3 and 8 TACR3* variants found in 9 propositi from a cohort of 173 normosmic CHH evaluated at Bicêtre Hospital.
Figure 2.
Molecular characterization, functional consequences and modeling of the p.Tyr267Asn TACR3 mutation.
Panel A. Evolutionary conservation of Tyr267. Tyr267 is perfectly conserved among NK3R orthologs and paralogs. The substitution is indicated below. Panel B. Modeling of the transmembrane region of NK3R. The tyrosine 267 and its substitution by an asparagine are pointed at the lipid bilayer. This position is extremely unfavorable for a polar residue such as asparagine. Panel C. Subcellular localization of ectopically expressed NK3R and Y267N mutant in non-permeabilized and permeabilized cells. Cells were transfected with the indicated expression vector and then treated for indirect immunofluorescence as described in the Methods section. The nuclei are counterstained by DAPI (blue). Upper panel: Z-stack projection of NK3R distribution in non-permeabilized cells obtained by confocal microscopy. Lower panel: fluorescence micrographs of fixed and permeabilized cells. Note the absence of Y267N NK3R mutant at the membrane (upper) despite its efficient expression in the cell (lower) whereas wild-type NK3R is localized at the plasma membrane. Panel D. NKB dose response of the reporter luc2P/SRE. Increasing concentrations of NKB led to an increase in the luciferase activity of wild-type NK3R (black circles). The mutant NK3R (red triangles) did not significantly enhance luciferase activity.
Figure 3.
Secretory free alpha-subunit (FAS) and gonadotropin responses to pulsatile GnRH administration.
Panel A. FAS concentrations in a man with nCHH caused by a homozygous TAC3 c.209-1G>C mutation, before and on day 19 of pulsatile GnRH administration (patient reported in part in [3]). Arrows indicate exogenous GnRH boluses (7 µg). Panel B. FAS concentration in a woman with nCHH caused by a mutation in TACR3 (subject II-6 in family I, Fig. 1A), before and on day 13 of pulsatile GnRH administration. Arrows indicate exogenous GnRH boluses (5 µg). Asterixes denote detectable FAS pulses, using Thomas' algorithm [24]. See Patients and Methods for details. Panel C. Pattern of gonadotropin secretion in a woman with complete nCHH and TACR3 mutation (subject II-6 in family I, Fig. 1A). Basal LH concentration was very low and FSH concentration was in the normal range. Panel D. LH pulsatility in this woman was restored by pulsatile GnRH administration, and the serum FSH level fell slightly during pulsatile GnRH administration. Serum estradiol (E2) and inhibin B (IB) levels before and after GnRH administration are indicated respectively at the top of panel C and D.
Table 2.
Characteristics of nCHH patients with biallelic TACR3 or TAC3 deleterious mutations selected for FSH/LH ratio calculation.
Figure 4.
FSH/LH ratio in 11 patients with nCHH caused by biallelic TAC3/TACR3 mutations.
Panel A. Significantly higher serum FSH/ serum LH ratios in 11 patients with nCHH caused by TAC3/TACR3 biallelic mutations than in patients with other genetic forms of CHH or in CHH patients with no mutation found in known genes. Note the Log scale on Y axis. A whole variance analysis by Kruskal-Wallis test (p<0.0001) was performed followed by post-hoc Newman-Keuls multiple comparison test; *indicates a significant difference between 2 groups (p<0.001). The threshold separating FSH/LH ratio in TAC3/TACR3 mutated nCHH subjects from those of patients with other genetic forms of CHH is indicated by an horizontal line. Panel B. Decrease in the FSH/LH ratio in one patient with TAC3 and two patients with TACR3 mutations (see Patients) during pulsatile GnRH administration. Note the Log scale on Y axis.