Loss-of-function mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism

Abstract

Gonadotropin-releasing hormone (GnRH) deficiency in the human presents either as normosmic idiopathic hypogonadotropic hypogonadism (nIHH) or with anosmia [Kallmann syndrome (KS)]. To date, several loci have been identified to cause these disorders, but only 30% of cases exhibit mutations in known genes. Recently, murine studies have demonstrated a critical role of the prokineticin pathway in olfactory bulb morphogenesis and GnRH secretion. Therefore, we hypothesize that mutations in prokineticin 2 (PROK2) underlie some cases of KS in humans and that animals deficient in Prok2 would be hypogonadotropic. One hundred IHH probands (50 nIHH and 50 KS) with no known mutations were examined for mutations in the PROK2 gene. Mutant PROK2s were examined in functional studies, and the reproductive phenotype of the Prok2(-/-) mice was also investigated. Two brothers with KS and their sister with nIHH harbored a homozygous deletion in the PROK2 gene (p.[I55fsX1]+[I55fsX1]). Another asymptomatic brother was heterozygous for the deletion, whereas both parents (deceased) had normal reproductive histories. The identified deletion results in a truncated PROK2 protein of 27 amino acids (rather than 81 in its mature form) that lacks bioactivity. In addition, Prok2(-/-) mice with olfactory bulb defects exhibited disrupted GnRH neuron migration, resulting in a dramatic decrease in GnRH neuron population in the hypothalamus as well as hypogonadotropic hypogonadism. Homozygous loss-of-function PROK2 mutations cause both KS and nIHH.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

IHH pedigree with a homozygous loss-of-function PROK2 mutation. (a) The pedigree reveals a family with two affected KS brothers (II-02 and II-03) and a normosmic IHH sister (II-05) carrying a homozygous deletion in PROK2 (p.[I55fsX1]+ [I55fsX1]). The unaffected brother (II-06) is heterozygous for the mutation. The proband is identified by the arrow. ○, females; □, males. Phenotypes are as described in the text. (b) Sequence electropherograms of WT and affected individuals for the homozygous PROK2 deletion. (c) Alignment of the truncated form of PROK2 (hRPOK2–27AA) with mature PROK2 (hPROK2). (d) In vitro analysis of the activity of the truncated hPROK2-27AA (open squares) and the mature PROK2 on a CHO cell line that stably expressed PROKR2 (open circles). No activation activity of the truncated hPROK2-27AA was observed at any of the tested concentrations.

Fig. 2.

Hypoplasia of the reproductive organs in Prok2−/− mice. (a and b) Macroscopic view of the testis, ovary, and uterus of 12-week-old Prok2−/− and WT mice. The reproductive organs of both male and female prok2−/− mice were small. (Scale bars: 5 mm.) (c) H&E-stained sections of testis from 12-week-old Prok2−/− and WT mice. Low magnification showed smaller seminiferous tubule in Prok2−/− mice. Higher magnification (rectangle in Upper Left) revealed no sperm in Prok2−/− mice. (Scale bars: 100 μm for low magnification and 25 μm for high magnification.) (d) H&E-stained sections of ovary from 12-week-old Prok2−/− and WT mice. Higher magnification (rectangle in the lower magnification) showed undeveloped follicles in Prok2−/− mice. (Scale bars: 200 μm for low magnification and 50 μm for high magnification.)

Fig. 3.

Abnormalities of the GnRH neurons in Prok2−/− mice. (a and b) Immunohistochemical staining of the preoptic region (a) and immunofluorescence staining of the median eminence (b) of 12-week-old WT and Prok2−/− mice. Fewer GnRH neurons were observed in Prok2−/− mice. The arrows in a indicate the cell bodies of GnRH-positive neurons; the arrows in b indicate the GnRH-immunoreactive axons. (Scale bars, 100 μm.) (c) Quantification of GnRH-positive neurons in the preoptic area of WT and Prok2−/− mice (**, P < 0.001; Student's t test; n = 4). (d and e) Measurement of LH and FSH hormones in age-matched WT and Prok2−/− mice. Both LH and FSH in male Prok2−/− were significantly lower than in WT control (*, P < 0.05; ***, P < 0.0001; Student's t test; n = 4).

Fig. 4.

prokr2 expression in the brain. Digoxigenin-labeled in situ hybridization of prokr2 in E13.5 (a–c) and adult (d and e) mouse brain. (a–c) In E13.5, prokr2-expressing cells were observed in the vomeronasal organ (high magnification in b) and in the basal forebrain (high magnification in c). (Scale bars: a, 250 μm; b, 50 μm; c, 25 μm.) In the adult (d and e), several prokr2-expressing cells were observed in the preoptic area. (e) High magnification of the rectangle in d. (Scale bars: d, 100 μm; e, 25 μm.)

Fig. 5.

prokr2 does not colocalize with GnRH. prokr2 expression was revealed by in situ hybridization (green), and GnRH was revealed by immunostaining (red) in the vomeronasal organ (A–C), in the basal forebrain (D–F) of E13.5, and in the preoptic area of adult mouse brain (G–I). (Scale bar: 50 μm.)

Fig. 6.

GnRH neurons were stalled in an exogenous spherical structure in the Prok2−/− mice. Migrating GnRH neurons passed the OB through their migration route at E13.5, E16.5, and E18.5 (a–c). In Prok2−/− mice, an exogenous spherical structure (asterisks in d–f) appeared between the two olfactory bulbs. GnRH neurons were stalled in the structure. (Scale bar: 100 μm.)