Impaired FGF signaling contributes to cleft lip and palate Bridget M. Riley*, M. Adela Mansilla*, Jinghong Ma†, Sandra Daack-Hirsch*, Brion S. Maher‡, Lisa M. Raffensperger*, Erilynn T. Russo*, Alexandre R. Vieira‡, Catherine Dode´ §, Moosa Mohammadi†, Mary L. Marazita‡¶, and Jeffrey C. Murray*ʈ *Department of Pediatrics, University of Iowa, Iowa City, IA 52242; †Department of Pharmacology, New York University School of Medicine, New York, NY 10016; ‡Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA 15219; §Institut Cochin et Laboratoire de Biochimie et Ge´ne´ tique Mole´culaire, Hoˆpital Cochin, 75014 Paris, France; and ¶Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15219 Edited by Mary-Claire King, University of Washington, Seattle, WA, and approved January 18, 2007 (received for review September 12, 2006) Nonsyndromic cleft lip and palate (NS CLP) is a complex birth defect plasia syndromes (FGFR1–FGFR3) and Kallmann syndrome resulting from a combination of genetic and environmental factors. (KS) (FGFR1). Cleft palate occurs in 44% of cases of Apert Several members of the FGF and FGFR families are expressed syndrome, which is characterized by combined craniosynostosis during craniofacial development and can rarely harbor mutations and syndactyly (12, 13). Two mutations in the FGFR2 gene, that result in human clefting syndromes. We hypothesized that S252W and P253R, account for almost all cases of Apert disruptions in this pathway might also contribute to NS CLP. We syndrome; the clefting phenotype is more commonly associated sequenced the coding regions and performed association testing with the S252W mutation (59%) than with the P253R mutation on 12 genes (FGFR1, FGFR2, FGFR3, FGF2, FGF3, FGF4, FGF7, FGF8, (17%) (12, 14). The autosomal-dominant KS, whose main fea- FGF9, FGF10, FGF18, and NUDT6) and used protein structure anal- tures are anosmia and hypogonadotrophic hypogonadism, is yses to predict the function of amino acid variants. Seven likely caused by loss-of-function mutations in FGFR1, and 5–10% of disease-causing mutations were identified, including: one non- these patients have a cleft (unpublished estimate) (15–17). sense mutation (R609X) in FGFR1, a de novo missense mutation Additionally, mutations in FGF10 are associated with autoso- (D73H) in FGF8, and other missense variants in FGFR1, FGFR2, and mal-dominant aplasia of lacrimal and salivary glands (18). FGFR3. Structural analysis of FGFR1, FGFR2, and FGF8 variants Animal models also support the involvement of FGFs and suggests that these mutations would impair the function of the FGFRs in the pathogenesis of CLP. Fgf10Ϫ/Ϫ (19), Fgfr2bϪ/Ϫ proteins, albeit through different mechanisms. Genotyping of SNPs (20), Fgf18Ϫ/Ϫ (21, 22), and Fgfr1 hypomorphic mice have cleft in the genes found associations between NS CLP and SNPs in FGF3, palate (23). Hypomorphic Fgf8neo/Ϫ mutant mice have cranio- FGF7, FGF10, FGF18, and FGFR1. The data suggest that the FGF facial defects, including abnormal development of the palate and signaling pathway may contribute to as much as 3–5% of NS CLP palatine bones (24). Moreover, disruption of the directional and will be a consideration in the clinical management of CLP. epithelial-mesenchymal Fgf10-Fgfr2b signaling has been found to result in cleft palate in mice (19). In this report, we provide fibroblast growth factor ͉ fibroblast growth factor receptor ͉ direct sequencing and association data showing that disruptions single-nucleotide polymorphism ͉ cleft palate in FGF signaling also contribute to CLP in humans. Results solated or nonsyndromic cleft lip and palate (NS CLP) is a Ϸ Icommon congenital anomaly affecting Ϸ1/700 live births Sequencing. Overall, 84 amplicons encoding 33,000 base pairs worldwide, although prevalence varies widely based on geo- were sequenced for the 12 FGF and FGFR genes in genomic graphic and socioeconomic status (1, 2). Significantly increased DNA for 184 individuals with NS CLP [see supporting infor- lifetime mortality ratios of 1.4 for males and 1.8 for females are mation (SI) Table 3]. Thirty-seven point mutations were iden- associated with NS CLP (3). Additionally, clefting requires tified in the exons or at the intron–exon junctions of the FGF and extensive medical interventions that create significant economic FGFR genes examined in families with NS CLP from both Iowa and social burdens for family and society (4). (16) and the Philippines (20). All mutations appear in SI Table Both genetic and environmental factors are known to play a 4, and the nine missense and nonsense mutations are listed in role in the development of NS CLP, complicating the identifi- Table 1. SI Table 5 contains the predictions from exon splice- cation of causal genes. A study examining recurrence patterns of enhancer programs on the effects of the synonymous mutations NS CLP estimated that as many as 14 interacting loci may be identified in the coding regions. None of these mutations has involved in clefting (5). Prior sequencing analysis of DNA been previously reported in the SNP database (dbSNP). When samples from NS CLP cases has indicated minor roles for we excluded from the analysis the initial ascertainment cases and mutations in MSX1, FOXE1, GLI2, MSX2, SKI, and SPRY2 in the etiology of NS CLP (6–8). Additionally, polymorphisms in Author contributions: B.M.R. and J.C.M. designed research; B.M.R., M.A.M., J.M., S.D.-H., the IRF6 gene have been found to be strongly associated with NS B.S.M., L.M.R., E.T.R., A.R.V., M.M., and M.L.M. performed research; C.D. contributed new CLP, accounting for Ϸ12% of clefting (9). reagents/analytic tools; B.M.R., M.A.M., B.S.M., L.M.R., E.T.R., A.R.V., M.M., M.L.M., and Mammalian fibroblast growth factors (FGF1–FGF10 and J.C.M. analyzed data; and B.M.R., M.M., M.L.M., and J.C.M. wrote the paper;. FGF16–FGF23) control a wide spectrum of biological functions The authors declare no conflict of interest. during development and adult life (10). The biological activities This article is a PNAS direct submission. of FGFs are conveyed by seven principal FGF receptor tyrosine Freely available online through the PNAS open access option. kinases encoded by four distinct genes (FGFR1–FGFR4). In the Abbreviations: FGFR, fibroblast growth factor receptor; KS, Kallmann syndrome; NS CLP, presence of heparan sulfate proteoglycans, two FGFs bind to two nonsyndromic cleft lip and palate. FGFRs, inducing receptor dimerization and enabling the intra- ʈTo whom correspondence should be addressed: Email: [email protected]. cellular tyrosine kinase domains to transphosphorylate and This article contains supporting information online at www.pnas.org/cgi/content/full/ become activated (11). Human diseases that arise from dysregu- 0607956104/DC1. lated FGF signaling include craniosynostosis and skeletal dys- © 2007 by The National Academy of Sciences of the USA 4512–4517 ͉ PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607956104 Downloaded by guest on September 26, 2021 Table 1. Missense and nonsense mutations in FGF pathway genes identified in NS CLP patients Gene Mutation Population Phenotype Frequency Control frequency UCSC browser position* FGFR1 M369I Philippine CPO UL CLP 2/1117 0/1368 Chr 8: 38,396,385 FGFR1 E467K Philippine UL CLP 2/1117 0/1384 Chr 8: 38,394,934 FGFR1 R609X Iowa UL CLP BL CLP 1/90 0/91 Chr 8: 38,392,574 FGFR2 D138N Philippine UL CLP BL CLP 5/1027 0/1392 Chr 10: 123,314,048 FGFR2 R84S Iowa BL CLP 1/362 0/1328 Chr 10: 123,315,066 FGFR3 V329I Philippine UL CL UL CLP BL CLP 8/977 5/961 Chr 4: 1,772,704 FGF8 D73H (de novo) Iowa BL CLP 1/262 0/1110 Chr 10: 103,524,599 FGF10 S59F Philippine BL CLP 2/91 3/83 Chr 5: 44,424,366 NUDT6 K172N Iowa Untyped CLP 1/87 0/89 Chr 4: 124,1276,421 CPO, cleft palate only; UL, unilateral; BL, bilateral; CL, cleft lip; CLP, cleft lip and palate; Untyped, cleft lip laterality unknown. *UCSC browser position from the May 2004 assembly. did a formal case-control study looking for the presence of maps to the intracellular juxtamembrane domain that links the M369I, E467K, R84S, D138N, V329I, and D73H, we found a transmembrane helix to the tyrosine kinase domain of FGFR1. significant difference between mutations in cases versus controls Both the M369 and E467 are conserved among the human, (P ϭ 0.04). The protein sequence-altering mutations are more chimp, mouse, rat, and chicken protein sequences (Fig. 2B). In common in cases of NS SLP than in controls. the crystal structure of the FGFR1 kinase domain, E467 is Two FGFR1 mutations, M369I and E467K, were identified in completely solvent-exposed and does not participate in the pedigrees from the Philippines. The M369I mutation was found overall folding of the FGFR1 kinase domain (Fig. 2A). Inter- in two unrelated families (SI Fig. 6). One family had three estingly, however, E467 is located four residues C terminal to affected siblings, as well as one unaffected sibling and an Y463, one of the main tyrosine phosphorylation sites of FGFR1. unaffected mother, each with the mutation. The father was Phosphorylated Y463 has been implicated in the binding and unavailable, but a paternal brother with a cleft did not have the phosphorylation of the SH2 containing signaling molecules such mutation. The E467K mutation was also found in cases from two as Crk and is part of the recognition sequence Y-E-L-P, a unrelated families (SI Fig. 7). In one family, the E467K was preferred binding motif for Crk SH2 domain (26, 27).