Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism

Abstract

Context: Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome.

Aim: To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH).

Patients and methods: We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin.

Results: A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed.

Conclusion: A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

Figure 1

(A) Nucleotide sequence of NDN gene showing the wild-type and the heterozygous variant identified in the male proband with Kallmann syndrome. (B) Pedigree of the family carrying the p.V318A variant in the necdin. The proband is identified by an arrow. Squares denote male subjects and circles denote female subjects. NDN and FGFR1 genotypes are shown in the family members who were studied. (C) Top, schematic diagram of necdin. Residue numbers correspond to human necdin. Bottom, amino acid sequence alignment of the necdin carboxyl terminus among mammalian species. The p.V318A patient mutation is indicated by an arrow.

Figure 2

Effects of wild-type or variant (p.V322A) mouse Ndn on GNRH expression. (A) GT1-7 mature GNRH neuronal cells were transiently co-transfected with 400 ng of reporter plasmids containing GNRH regulatory elements driving luciferase, as indicated by schematic diagrams, with 200 ng of either empty expression vector, wild-type Ndn expression plasmid, or p.V322A Ndn expression plasmid. (B) Dosage effect of wild-type Ndn or p.V322A Ndn on GNRH expression. GT1-7 cells were transiently transfected with 400 ng GNRH-E1/GNRH-P luciferase reporter plasmid with increasing amounts (50, 75, 100, and 200 ng) of either wild-type Ndn or variant p.V322A Ndn expression plasmid. Luciferase values were normalized to a co-transfected β-galactosidase reporter to control for transfection efficiency. Data are presented as fold luciferase/β-galactosidase activity, relative to empty expression vector control, mean±S.D., of four independent experiments. *Significantly different from empty vector by Student’s t-test (P<0.05). E1, GNRH enhancer-1; E2, GNRH enhancer-2; E3, GNRH enhancer-3; GNRH-P, GNRH promoter.