Expansion of the complex genotypic and phenotypic spectrum of FGFR2-associated neurocutaneous syndromes

Abstract

The fibroblast growth factor receptors comprise a family of related but individually distinct tyrosine kinase receptors. Within this family, FGFR2 is a key regulator in many biological processes, e.g., cell proliferation, tumorigenesis, metastasis, and angiogenesis. Heterozygous activating non-mosaic germline variants in FGFR2 have been linked to numerous autosomal dominantly inherited disorders including several craniosynostoses and skeletal dysplasia syndromes. We report on a girl with cutaneous nevi, ocular malformations, macrocephaly, mild developmental delay, and the initial clinical diagnosis of Schimmelpenning-Feuerstein-Mims syndrome, a very rare mosaic neurocutaneous disorder caused by postzygotic missense variants in HRAS, KRAS, and NRAS. Exome sequencing of blood and affected skin tissue identified the mosaic variant c.1647=/T > G p.(Asn549=/Lys) in FGFR2, upstream of the RAS signaling pathway. The variant is located in the tyrosine kinase domain of FGFR2 in a region that regulates the activity of the receptor and structural mapping and functional characterization revealed that it results in constitutive receptor activation. Overall, our findings indicate FGFR2-associated neurocutaneous syndrome as the accurate clinical-molecular diagnosis for the reported individual, and thereby expand the complex genotypic and phenotypic spectrum of FGFR-associated disorders. We conclude that molecular analysis of FGFR2 should be considered in the genetic workup of individuals with the clinical suspicion of a mosaic neurocutaneous condition, as the knowledge of the molecular cause might have relevant implications for genetic counseling, prognosis, tumor surveillance and potential treatment options.

Fig. 1

Clinical pictures and cranial images of the affected individual. A–C Clinical characteristics of the affected girl at the age of 2 years and 9 months included widespread nevus sebaceous on the face affecting the right eyebrow and areas of hair loss on the scalp, hypertelorism and low-set ears. D Hairless areas on the head and patchy skin pigmentation restricted to the right side of her neck (indicated by black arrow). E Linear epidermal nevi restricted to the left side of the abdomen (indicated by black arrow). F Widespread epidermal nevi on the torso. G Broad great toes on both sides. H Cranial T1 weighted mid-sagittal MRI (with gadolinium) at the age of 6 months depicting abnormal shape of the skull and a hypoplastic midface. Skull base appears dysmorphic with a steep clivus, a shallow sella and an enlarged posterior fossa. I Cranial T1 weighted axial MRI shows slightly widened internal cerebrospinal fluid spaces

Fig. 2

Molecular findings. Deep sequencing analysis was used to validate the exome data and confirmed the mosaic FGFR2 variant c.1647=/T > G p.(Asn549=/Lys) in DNA extracted from biopsy tissue. The variant was present in 32% (45,352/139,782) of the reads from the FFPE sample (A), in 12% (52,451/450,905) of the fibroblasts (B) and was not (only 0.15%) detectable in DNA extracted from peripheral blood lymphocytes (545/366,227) (C)

Fig. 3

Overview of the RAS-MAPK signaling pathway, location and functional characterization of the p.(Asn549Lys) variant in FGFR2. A Schematic overview of the FGFR2 receptor. Red arrow points to the location of the variant identified in this study. TK1, tyrosine kinase domain 1; TK2, tyrosine kinase domain 2. Selected previously reported variants causing different FGFR2-associated disorders and their location are indicated by grey arrows. BSTV syndrome, Beare–Stevenson cutis gyrata syndrome. LADD syndrome, lacrimoauriculodentodigital syndrome. B Postzygotic activating variants in HRAS, KRAS and NRAS (indicated in red) represent known causes of SFM syndrome. The identified mosaic variant reported here leads to a constitutive activation of fibroblast growth factor receptor 2 (FGFR2) upstream of the RAS signaling cascade. C HEK293 cells were transiently transfected with vectors containing cDNA for FLAG-tagged wild-type FGFR2 (WT) or FGFR2 harboring the identified variant c.1647T > G p.(Asn549Lys). Untransfected cells served as negative control (−). Western blot analysis of FGRF2 expression revealed equal protein expression of wild-type and p.(Asn549Lys) FGFR2 (upper panel). Equal protein loading was confirmed by re-probing of the membrane with antibodies against actin (lower panel). D Representative Western blot showing increased tyrosine kinase activity of FGFR2 carrying the p.(Asn549Lys) amino acid substitution compared to wild-type FGFR2. HEK293 cells were transiently transfected with FLAG-tagged wild-type FGFR2, FGFR2 p.(Asn549Lys), or left untransfected as control (–). Lysates from cells were subjected to immunoprecipitation (IP) with anti-FLAG antibodies followed by SDS-PAGE and immunoblotting with anti-p-Tyr antibodies. E Quantification of pFGFR2 levels in cells expressing wild-type FGRF2 (WT) or FGFR2 harboring the identified amino acid substitution p.(Asn549Lys). pFGFR2 signal was significantly higher in cells expressing the p.(Asn549Lys) FGFR2 variant (n = 3; mean ± SD)

Fig. 4

Structure of the FGFR2 kinase domain. A Crystal structure of the FGFR2 kinase domain (PDB: 2PVF) shown as cartoon. B–D The kinase hinge region regulating the kinase activation is shown enlarged, with important residues shown as sticks. B In the unphosphorylated wild-type structure of the FGFR2 kinase domain (PDB: 2PSQ) the amino acids Ile547, Asn549, Glu565 and Lys641 form a network of hydrogen bonds which serves as a molecular brake by inhibiting the auto-kinase activity of the FGFR2 receptor (Chen et al. 2007). C In the phosphorylated wild-type structure (PDB: 2PVF), this molecular brake is disengaged, resulting in kinase activation (Chen et al. 2007). D The variant p.(Asn549Lys) identified in this study compromises the molecular brake in the unphosphorylated state