Loss-of-function variants in SEMA3F and PLXNA3 encoding semaphorin-3F and its receptor plexin-A3 respectively cause idiopathic hypogonadotropic hypogonadism

Abstract

Purpose: Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by absent puberty and subsequent infertility due to gonadotropin-releasing hormone (GnRH) deficiency. IHH can be accompanied by normal or compromised olfaction (Kallmann syndrome). Several semaphorins are known potent modulators of GnRH, olfactory, and vomeronasal system development. In this study, we investigated the role of Semaphorin-3F signaling in the etiology of IHH.

Methods: We screened 216 IHH patients by exome sequencing. We transiently transfected HEK293T cells with plasmids encoding wild type (WT) or corresponding variants to investigate the functional consequences. We performed fluorescent IHC to assess SEMA3F and PLXNA3 expression both in the nasal region and at the nasal/forebrain junction during the early human fetal development.

Results: We identified ten rare missense variants in SEMA3F and PLXNA3 in 15 patients from 11 independent families. Most of these variants were predicted to be deleterious by functional assays. SEMA3F and PLXNA3 are both expressed along the olfactory nerve and intracranial projection of the vomeronasal nerve/terminal nerve. PLXNA1-A3 are expressed in the early migratory GnRH neurons.

Conclusion: SEMA3F signaling through PLXNA1-A3 is involved in the guidance of GnRH neurons and of olfactory and vomeronasal nerve fibers in humans. Overall, our findings suggest that Semaphorin-3F signaling insufficiency contributes to the pathogenesis of IHH.

Figure 1. SEMA3F Mutations in the etiology of idiopathic hypogonadotropic hypogonadism.

A. The mutations are depicted on the functional gene diagram of SEMA3F. B. The pedigrees of seven families with SEMA3F mutations are shown. Affected males and females are represented by black squares and black circles respectively. White square symbols indicate unaffected male family members, white circle symbols represent unaffected female family members, and the double line indicates consanguinity. Under each symbol are the genotypes in the same order as the gene and variantdescriptions, with WT and M denoting wild type and mutant, respectively. ND: Not determined. C. The T2-weighted magnetic resonance imaging (MRI) from a healthy control (a) and from patient DII-2 (b). The arrows point to normal (a) and aplastic olfactory bulbs (b).

Figure 2. PLXNA3 mutations in patients with idiopathic hypogonadotropic hypogonadism.

A. The diagram of PLXNA3 showing the positions of the missense mutations found in patients. B. The pedigrees of the three families with PLXNA3 mutations are shown. Note that patients in Family G in Figure 1 also have PLXNA3 mutations in addition to SEMA3F mutations. Affected males and females are represented by black squares and black circles respectively. White square symbols indicate unaffected male family members, white circle symbols represent unaffected female family members, and the double line indicates consanguinity. Under each symbol are the genotypes in the same order as the gene and variantdescriptions, with WT and M denoting wild type and mutant, respectively.

Figure 3. Semaphorin 3F and Plexin A3 are expressed along the migratory route of GnRH neurons in human fetuses.

Fluorescent IHC was performed to assess SEMA3F and PLXNA3 expression both in the nasal region (A) and at the nasal/forebrain junction (I) during the early fetal development. (B-D) Representative immunostaining for GnRH, Peripherin and Plexin A3 on sagittal sections of a GW7.5 human fetus. (E-H) Magnification of the boxed area in B, revealing that PLXNA3 is found in the peripherin-positive olfactory/vomeronasal scaffold, as well as in GnRH neurons (arrows) during their migration through the nasal compartment. (J-L) Representative pictures of immunolabelings for GnRH, PLXNA3 and SEMA3F on coronal sections of a GW10.5 human fetus. (M-P) Magnification of the boxed area in J. The olfactory and vomeronasal/terminal nerves still show immunoreactivity for PLXNA3 at the nose/forebrain junction. SEMA3F is strongly expressed in this region and detected along the PLXNA3-positive nerves. GnRH neurons migrating inside the forebrain do not longer express PLXNA3. fb, forebrain; ob, olfactory bulbs; oe, olfactory epithelium; on, olfactory nerves; vnn/tn, vomeronasal/terminal nerves. Scale bars: B, J = 500 μm; E, M = 10 μm.

Figure 4. PLXNA1 and PLXNA2 could also contribute to SEMA3F signaling in the human fetal nose.

Since SEMA3F can signal not only through PLXNA3, but through the PLXNA1-A4 family, additional stainings were performed to check for PLXNA1-A4 expression in the nasal compartment of a GW7.5 human fetus. In addition to PLXNA3, migrating GnRH neurons and the olfactory/vomeronasal scaffold also express PLXNA1 (C-D) while PLXNA2 was detected in GnRH neurons and other cells of the migratory mass, but not in the olfactory/vomeronasal projections (E-F). However, PLXNA4 immunoreactivity was only found in scattered cells, distant from the migratory route of GNRH neurons (G-H). ob, olfactory bulbs. Scale bars: B = 500 μm; E,C,G = 10 μm.