Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 are identified in individuals with congenital hypogonadotropic hypogonadism

Abstract

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ~12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH.

Figure 1

FGF-Network-Associated Genes Harbor Mutations in CHH Individuals (A) A simplified schematic of the FGF pathway includes frequencies of mutations identified in CHH individuals. KAL1 and FLRT3, encoding enhancers of FGF signaling, are shown in green; IL17RD, DUSP6, and SPRY4, encoding inhibitors, are in red; HS6ST1, encoding the HS-modifying enzyme, is in blue; FGF8 and FGF17, encoding ligands, are in yellow; and FGFR1, encoding the receptor FGFR1, is in gray. (B) Number of nIHH and KS individuals with mutations in each gene.

Figure 2

An Integrated FGF Signaling Network of Protein-Protein Interactions and Gene-Synexpression Data Implicated in Human Reproduction Proteins identified by IBAS to significantly interact with FGF8 and FGFR1 are shown in this network. On the basis of high-quality protein-protein interaction data from InWeb, 17 proteins have genome-wide-significant IBAS scores as a result of their interaction patterns with the FGFR1-FGF8 receptor-ligand pair. Moreover, five proteins (FGF17, IL17RD, DUSP6, SPRY4, and FLRT3) are synexpressed with FGF8 and FGFR1 during development. IL17RD and FGF17 are the top-scoring candidates among 12,507 proteins in InWeb.

Figure 3

IL17RD Mutations in Individuals with CHH (A) Schematic of IL17RD domain structure and location of alterations. Abbreviations are as follows: SP, signal peptide; Ig-like, immunoglobulin-like; TM, transmembrane; SEFIR, SEF/IL-17R domain; and ^*, homozygous. (B) Pedigrees of KS probands harboring mutations in IL17RD. “F” indicates family number. The proband of each family is indicated by an arrow and “P.” The available genotypes are indicated below each individual. Numbers within symbols denote the number of additional siblings. Squares depict males, and circles depict females. (C) Transcription reporter assay of WT and altered IL17RD. HEK293 cells were transiently cotransfected with FGFR1c-encoding cDNA and WT or altered IL17RD together with AP-1 luciferase reporter and then stimulated with FGF8. Plotted are the means ± SEM of five independent experiments run in quadruplet; the results have been normalized to EV-treated cells (100%). The p.Lys131Thr, p.Pro306Ser, and p.Ser468Leu altered proteins showed loss of function in this assay. The p.Tyr379Cys and p.Pro577Gln altered proteins also demonstrated reduced inhibition activity, which was borderline significant (p = 0.06). p.Lys162Arg and p.Ala735Val were similar to the WT in this assay. Δ326, a truncated IL17RD lacking the intracellular domain (which is the domain that interacts with FGFR1) served as a negative control. Abbreviations are as follows: a, p < 0.05; b, p = 0.06; and ns, not significant. (D) Widefield epifluorescence images of IL17RD immunoreactivity (red) and nuclear marker DAPI (blue) in the nasal region of E10.5 mice (sagittal view) show IL17RD localization in the epithelium of the olfactory placode (left panel). Scale bars represent 100 μm (left panel) and 10 μm (right panel). The area in the white box is enlarged in the right panel. Abbreviations are as follows: MES, mesenchyme; Opd, olfactory placode (dorsal); Opv, olfactory placode (ventral); c, caudal; d, dorsal; r, rostral; and v, ventral; and Sef, similar expression as FGF (former name of IL17RD). (E) Widefield epifluorescence images of IL17RD immunoreactivity (red) and nuclear marker DAPI (blue) in E12.5 mouse ventral forebrain (sagittal view) of WT (left) and homozygous Fgf8 hypomorphic (right) embryos illustrate the complete absence of IL17RD in Fgf8 hypomorphic embryos. IL17RD immunoreactivity was high in the septal area and in the midbrain-hindbrain junction of the WT embryo (upper inset) and nearly undetectable in the olfactory epithelium, where migrating GnRH neurons (green) in the developing nasal regions are localized (lower inset). Scale bars represent 100 μm for all images except the insets, in which they represent 50 μm. Abbreviations are as follows: cb, cerebellum; GE, ganglionic eminence; SA, septal area; and oe, olfactory epithelium; and Sef, similar expression as FGF (former name of IL17RD).

Figure 4

FGF17 Mutations in Individuals with CHH (A) In cells transfected with FGFR1 and a FGF luciferase reporter, increasing doses of WT FGF17 produce a typical sigmoidal dose-response curve (blue), which is abolished by the FGFR1c p.Leu342Ser alteration. (B) Schematic of FGF17 structural domains and location of the identified alterations. The following abbreviations is used: SP, signal peptide. (C) The interface between FGF8b (orange) and D2 and D2-D3 linker (green) of FGFR2c as observed in the FGF8b-FGFR2c crystal structure (Protein Data Bank ID 2FDB³⁷) depicts the hydrogen bond between Arg177 (R177) of FGF8b and Asp247 (D247) in D2 of FGFR2c and the hydrogen bonds between FGF8b and FGFR Arg251 (R251). (D) The hydrophobic interior of the β-trefoil core of FGF8b illustrates the hydrophobic interactions among Ile108 (I108), Met151 (M151), Val65 (V65), Val67 (V67), Val99 (V99) (Ile99, I99 in FGF17), Leu86 (L86), Ile73 (I73), and Phe129 (F129). (E) SPR analysis of the binding of FGF17^WT (orange) and FGF17^p.Arg177His (green) to the FGFR1c ectodomain. FGF17^WT and FGF17^p.Arg177His were immobilized onto CM5 sensor chips, and varying concentrations of FGFR1c ectodomain were passed over. The full dose-response curves for FGF17^WT-FGFR1c and FGF17^p.Arg177His-FGFR1c binding were generated. For comparison, an overlay of the sensorgrams obtained upon injections of 800 nM solutions of FGFR1c over each ligand is shown. (F) FGF luciferase-reporter assay showing that the p.Arg177His (green) and p.Ile108Thr (red) FGF17 alterations impair the ligand’s biological activity. Abbreviations are as follows: ns, not significant; and a, p < 0.05. (G) The FGFR1c p.Arg250Gln and FGF17 p.Ile108Thr loss-of-function alterations act in additive fashion. Luciferase activity was plotted as the mean ± SEM of three independent experiments performed in triplicate (data are normalized to the WT), and a dose-response curve was fitted as described in Subjects and Methods. The following abbreviation is used: a, p < 0.05. (H) Fgf17 is expressed in the olfactory placode in a Fgf8-dependent manner. Fgf17 WM-ISH of E10.5 WT (panels 1 and 3) and Fgf8 homozygous hypomorphic (Fgf8^−/−; panels 2 and 4) embryos is shown. A 679 nt digoxigenin-labeled Fgf17 riboprobe was used. Panels 1 and 2 show sagittal views of embryonic heads; scale bars represent 500 mm. Panels 3 and 4 show ventral views of embryonic heads; scale bars represent 250 mm. Arrows are used as follows: solid arrows, midbrain-hindbrain junction; dotted arrows, commissural plate; and arrowheads, medial olfactory placode (where GnRH neurons emerge).

Figure 5

FGF-Network-Associated Genes Contribute to the Oligogenic Architecture of CHH (A) Pedigree of CHH-affected family with oligogenic inheritance. The proband is indicated by an arrow and “P.” The available genotypes are indicated below each individual. Numbers within symbols denote the number of additional siblings. Squares depict males, and circles depict females. (B) Percentages of individuals with monoallelic, biallelic, and oligogenic mutations in FGF-network-associated genes and/or other genes known to be mutated in CHH. (C) Number of alleles with mutations in CHH individuals and controls.