Functional consequences of AXL sequence variants in hypogonadotropic hypogonadism

Abstract

Context: Prior studies showed that Axl /Tyro3 null mice have delayed first estrus and abnormal cyclicity due to developmental defects in GnRH neuron migration and survival.

Objective: The objective of the study was to test whether the absence of Axl would alter reproductive function in mice and that mutations in AXL are present in patients with Kallmann syndrome (KS) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH).

Design and setting: The sexual maturation of Axl null mice was examined. The coding region of AXL was sequenced in 104 unrelated, carefully phenotyped KS or nIHH subjects. Frequency of mutations was compared with other causes of GnRH deficiency. Functional assays were performed on the detected mutations.

Results: Axl null mice demonstrated delay in first estrus and the interval between vaginal opening and first estrus. Three missense AXL mutations (p.L50F, p.S202C, and p.Q361P) and one intronic variant 6 bp upstream from the start of exon 5 (c.586-6 C>T) were identified in two KS and 2 two nIHH subjects. Comparison of the frequencies of AXL mutations with other putative causes of idiopathic hypogonadotropic hypogonadism confirmed they are rare variants. Testing of the c.586-6 C>T mutation revealed no abnormal splicing. Surface plasmon resonance analysis of the p.L50F, p.S202C, and p.Q361P mutations showed no altered Gas6 ligand binding. In contrast, GT1-7 GnRH neuronal cells expressing p.S202C or p.Q361P demonstrated defective ligand dependent receptor processing and importantly aberrant neuronal migration. In addition, the p.Q361P showed defective ligand independent chemotaxis.

Conclusions: Functional consequences of AXL sequence variants in patients with idiopathic hypogonadotropic hypogonadism support the importance of AXL and the Tyro3, Axl, Mer (TAM) family in reproductive development.

Figures

Figure 1.

Family pedigrees of probands identified with AXL mutations. Squares denote males and circles denote females. AXL mutation details are shown below each proband and phenotype details are presented in the text.

Figure 2.

A, Location of mutations within schema of AXL receptor molecule. B, The strategies to assess the effects of the mutations on Gas6 ligand binding, chemotaxis independent of ligand, receptor processing, and migration. K, tyrosine kinase; N, N terminus; C, C terminus.

Figure 3.

Functional characterization of mutant AXL residues roles on Gas6-dependent AXL processing. A, AXL cleavage was determined in GT1-7 GnRH neuronal cells expressing WT or mutant AXL-Fc protein. For the cleavage assay, whole-cell protein lysates or concentrated media supernatant were immunoblotted to detect intact and extracellular AXL (ECD) levels. B, Bar graphs depicting effect of Gas6 on the mean relative expression of cleaved AXL (ECD) to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vector, WT AXL, and mutant AXL overexpressing GT1-7 GnRH neuronal cells. Letters represent significant difference from each other (a, difference from vector control; b, difference from WT AXL overexpressing cells) (P < .05, n = 3).

Figure 4.

AXL mutation alters Gas6-induced GnRH neuronal migration and alters aggregation/chemotaxis in GT1-7 cells. A, GT1-7 cells overexpressing WT or mutant AXL were tested for functional effects on migration. Bar graph depicts effect of WT and mutant AXL overexpression (as shown in inset) on migration of GT1-7 neurons in the presence Gas6. The average number of cells that have migrated is shown and expressed as mean ± SEM, and letters represent significant difference from each other (a, difference from vector control and PBS treated group; b, difference from vector control and Gas6 treated group; c, difference from WT AXL overexpressing cells treated with Gas6) (P ≤ .05, two way ANOVA with post hoc Tukeys multiple comparisons test, n = 3). B, GT1-7 cells overexpressing WT or mutant AXL were tested for their aggregation ability. The number of clumps formed for each condition is shown. Letters represent significant difference from each other (a, difference from vector control; b, difference from WT AXL overexpressing cells) (n = 3). *, P ≤ .05.