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Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: characterization of neuroendocrine phenotypes and novel mutations.

Francou B, Bouligand J, Voican A, Amazit L, Trabado S, Fagart J, Meduri G, Brailly-Tabard S, Chanson P, Lecomte P, Guiochon-Mantel A, Young J - PLoS ONE (2011)

Bottom Line: We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2.Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio.The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, Faculté de Médecine Paris-Sud UMR-S693, Le Kremlin Bicêtre, France.

ABSTRACT

Context: TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway.

Objective: To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations.

Results: From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%). We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants) found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn) probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio.

Conclusion: The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations.

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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.
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pone-0025614-g002: 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.

Mentions: The Tyr267 residue is highly conserved among the three human tachykinin receptors and in all NK3R orthologs (Fig. 2A). Modeling was used to predict the potential impact of this point mutation on the three-dimensional organization of NK3R. We found that the hydrophobic Tyr267 residue is located in the middle of the fifth transmembrane segment and points towards the lipid bilayer (Fig. 2B). Introduction of an asparagine residue at position 267 (Y267N mutation) places a polar residue in a highly hydrophobic environment. Thus, it is likely that the asparagine side chain, in order to adopt a buried position, will induce transmembrane reorganization (twist or rotation). Therefore, the Tyr267Asn mutation leads probably to NK3R misfolding and dysfunction.


Normosmic congenital hypogonadotropic hypogonadism due to TAC3/TACR3 mutations: characterization of neuroendocrine phenotypes and novel mutations.

Francou B, Bouligand J, Voican A, Amazit L, Trabado S, Fagart J, Meduri G, Brailly-Tabard S, Chanson P, Lecomte P, Guiochon-Mantel A, Young J - PLoS ONE (2011)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198730&req=5

pone-0025614-g002: 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.
Mentions: The Tyr267 residue is highly conserved among the three human tachykinin receptors and in all NK3R orthologs (Fig. 2A). Modeling was used to predict the potential impact of this point mutation on the three-dimensional organization of NK3R. We found that the hydrophobic Tyr267 residue is located in the middle of the fifth transmembrane segment and points towards the lipid bilayer (Fig. 2B). Introduction of an asparagine residue at position 267 (Y267N mutation) places a polar residue in a highly hydrophobic environment. Thus, it is likely that the asparagine side chain, in order to adopt a buried position, will induce transmembrane reorganization (twist or rotation). Therefore, the Tyr267Asn mutation leads probably to NK3R misfolding and dysfunction.

Bottom Line: We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2.Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio.The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, Faculté de Médecine Paris-Sud UMR-S693, Le Kremlin Bicêtre, France.

ABSTRACT

Context: TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway.

Objective: To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations.

Results: From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%). We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants) found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn) probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio.

Conclusion: The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations.

Show MeSH
Related in: MedlinePlus