Mutations in prokineticin 2 and prokineticin receptor 2 genes in human gonadotrophin-releasing hormone deficiency: molecular genetics and clinical spectrum

Abstract

Context: Mice deficient in prokineticin 2(PROK2) and prokineticin receptor2 (PROKR2) exhibit variable olfactory bulb dysgenesis and GnRH neuronal migration defects reminiscent of human GnRH deficiency.

Objectives: We aimed to screen a large cohort of patients with Kallmann syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (IHH) for mutations in PROK2/PROKR2, evaluate their prevalence, define the genotype/phenotype relationship, and assess the functionality of these mutant alleles in vitro.

Design: Sequencing of the PROK2 and PROKR2 genes was performed in 170 KS patients and 154 nIHH. Mutations were examined using early growth response 1-luciferase assays in HEK 293 cells and aequorin assays in Chinese hamster ovary cells.

Results: Four heterozygous and one homozygous PROK2 mutation (p.A24P, p.C34Y, p.I50M, p.R73C, and p.I55fsX1) were identified in five probands. Four probands had KS and one nIHH, and all had absent puberty. Each mutant peptide impaired receptor signaling in vitro except the I50M. There were 11 patients who carried a heterozygous PROKR2 mutation (p.R85C, p.Y113H, p.V115M, p.R164Q, p.L173R, p.W178S, p.S188L, p.R248Q, p.V331M, and p.R357W). Among them, six had KS, four nIHH, and one KS proband carried both a PROKR2 (p.V115M) and PROK2 (p.A24P) mutation. Reproductive phenotypes ranged from absent to partial puberty to complete reversal of GnRH deficiency after discontinuation of therapy. All mutant alleles appear to decrease intracellular calcium mobilization; seven exhibited decreased MAPK signaling, and six displayed decreased receptor expression. Nonreproductive phenotypes included fibrous dysplasia, sleep disorder, synkinesia, and epilepsy. Finally, considerable variability was evident in family members with the same mutation, including asymptomatic carriers.

Conclusion: Loss-of-function mutations in PROK2 and PROKR2 underlie both KS and nIHH.

Figure 1

PROK2 and PROKR2 mutations. A, Schematic of the PROK2 gene. Mutations identified in the present cohort are identified with an arrow. Exons 1, 2, and 4 comprise the regular form of PROK2 (81 amino acids); the long form splice variant contains exons 1–4 (102 amino acids). B, Schematic of predicted PROKR2 protein using the SOSUI secondary structure prediction program. Mutations identified in this study are shown in blue.

Figure 2

PROK2 mutants (C34Y, I55fsX, and R73C) are loss of function. CHO cells stably expressing WT PROKR2 and aequorin reporter were treated with increasing doses of WT or mutant PROK2, and intracellular Ca2+ influx was monitored via aequorin excitation. Data are plotted as mean ± se of three independent experiments. max, Maximum.

Figure 3

Functional and expression analysis of PROKR2 mutants. A and D, Activity of PROKR2 mutants in transcription assay. HEK293 cells were transiently transfected with Egr1-lucifarase reporter and PROKR2 mutant constructs, and luciferase activity was monitored at increasing doses of PROK2. B and E, Activity of PROKR2 mutants in Ca²⁺ influx assay. CHO cells stably expressing the photoprotein aequorin were transiently transfected with WT or mutant PROKR2 plasmids, and aequorin luminescence was recorded after stimulation with PROK2. For both assays, data are presented as means ± se of three independent experiments, expressed as percentage of maximal (max) response of WT PROKR2. C and F, Western analysis of PROKR2 mutants. HEK293 cells were transiently transfected with WT or mutant PROKR2 plasmids, and equal amounts of whole cell lysate were analyzed by reduced SDS-PAGE. PROKR2 expression was visualized using anti-V5 antibodies and normalized to β-actin expression. “Mutant: WT” denotes the ratio of protein expressed.

Figure 4

Variable neuroendocrine profiles in subjects with PROK2 and PROKR2 mutations. A, Representative neuroendocrine baselines in subject nos. 6, 7, 9, 10, 12, and 15 with absent puberty. Ts for the three male subjects were all less than 30 ng/dl; E₂ for subject no. 2 was less than 32 pg/ml. B, Neuroendocrine profile for subject no. 11. Baseline profiling reveals a hypergonadotropic LH secretion pattern in the setting of hypogonadal T. C, Neuroendocrine profile for female subject no. 14. Baseline profiling shows LH secretion that is elevated for her low-normal E₂ level.

Figure 5

Pedigree analysis reveals incomplete penetrance for PROK2 and PROKR2. Squares are males, and circles are females. The arrow denotes the proband. SB, Stillborn.