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Apparently synonymous substitutions in FGFR2affect splicing and result in mild Crouzon syndrome
© Fenwick et al.; licensee BioMed Central Ltd. 2014
Received: 9 June 2014
Accepted: 4 August 2014
Published: 31 August 2014
Mutations of fibroblast growth factor receptor 2 (FGFR2) account for a higher proportion of genetic cases of craniosynostosis than any other gene, and are associated with a wide spectrum of severity of clinical problems. Many of these mutations are highly recurrent and their associated features well documented. Crouzon syndrome is typically caused by heterozygous missense mutations in the third immunoglobulin domain of FGFR2.
Here we describe two families, each segregating a different, previously unreported FGFR2 mutation of the same nucleotide, c.1083A>G and c.1083A>T, both of which encode an apparently synonymous change at the Pro361 codon. We provide experimental evidence that these mutations affect normal FGFR2 splicing and document the clinical consequences, which include a mild Crouzon syndrome phenotype and reduced penetrance of craniosynostosis.
These observations add to a growing list of FGFR2 mutations that affect splicing and provide important clinical information for genetic counselling of families affected by these specific mutations.
KeywordsCraniosynostosis Crouzon syndrome Expressivity FGFR2 Penetrance Splicing Synonymous substitution
Craniosynostosis defines the premature fusion of the cranial sutures and has an overall prevalence of 1 in 2100-2300 live births ,. Nearly one quarter of craniosynostosis has a genetic aetiology ,; there is considerable genetic heterogeneity and frequent phenotypic overlap between different syndromes. Genes encoding three members of the fibroblast growth factor receptor family (FGFR1, FGFR2 and FGFR3) are commonly mutated in individuals with craniosynostosis. Heterozygous mutations in FGFR2, which are frequently recurrent, account for ~28% of genetic cases  and cause Crouzon ,, Pfeiffer -, Apert , Beare-Stevenson  and bent bone dysplasia  syndromes. All involve synostosis of the coronal and other cranial sutures, a distinctive “crouzonoid” craniofacial appearance (comprising hypertelorism, exorbitism, prominent nose, and midface hypoplasia), but differ in the presence and extent of abnormalities of the hands and feet, other skeletal manifestations and dermatological features ,.
FGFR2, like the other members of the FGFR family, comprises an extracellular ligand-binding region (composed of three immunoglobulin-like domains), a single transmembrane peptide and a cytoplasmic tyrosine kinase domain. Mutually exclusive alternative splicing of exons IIIb and IIIc gives rise to epithelial and mesenchymal isoforms (FGFR2b and FGFR2c) respectively . These alternative extracellular domains interact with different repertoires of fibroblast growth factors (FGFs) to regulate downstream processes such as proliferation, differentiation and cell migration .
Here we describe two families heterozygous for the same, previously unreported apparently synonymous variant in FGFR2 [p.(Pro361Pro)], although caused by differing nucleotide substitutions. The mutation carriers in both families exhibit features of mild Crouzon syndrome, and a minority required craniofacial surgery. We propose that this variant is in fact pathogenic and demonstrate the generation of abnormal cDNA products resulting from incorrect splicing of exon IIIc in the mutant allele. This finding highlights the challenges posed in interpreting such synonymous variants when providing genetic counselling for affected families.
Computed tomography (CT) of the skull of individual III-1 at the age of 2.8 years demonstrated right lambdoid and occipitomastoid synostosis, all other major cranial sutures being patent. Ophthalmological review identified slightly reduced visual acuity and a latent divergent squint with slight left hypophoria. The patient is now four years old and has not undergone any surgical intervention, as she has a good overall head shape with no major midface retrusion, is making good developmental progress, and has no features to suggest significant intracranial restriction.
The male proband (III-2 in Figure 1C), born after an uneventful pregnancy, was referred for craniofacial assessment at the age of three months. Physical examination showed a mild cloverleaf skull with temporal bulging and reduced OFC (36 cm; -2.2 SD), hypertelorism, and severe exorbitism mainly at the infra-orbital level. Skull X-ray and CT showed pansynostosis and multiple craniolacunae, with no intracerebral anomalies.
Owing to the severe peri-orbital features and the absence of deformations of the upper and lower extremities a clinical diagnosis of Crouzon syndrome was suggested. The patient's mother, grandmother and several cousins were reported to show mild facial features also suggestive of this diagnosis.
The proband underwent fronto-orbital advancement at the age of five months. Since the occiput was still severely flattened and both lambdoid sutures were fused, occipital craniotomy and remodelling was performed at the age of twelve months. Clumsiness and motor delay were first noted aged 18 months; psychological testing at the age of 12.8 years gave scores for non-verbal intelligence of 80 (SON-R) and visual-motor integration of 81. Clonidine was prescribed due to high distractibility and he underwent special education. During childhood, the exorbitism increased requiring further orbital advancement and cranial vault remodelling at the age of eight years. Several deciduous and permanent teeth were extracted because of Class III malocclusion and dental crowding.
Independently of these events, a second patient (III-1) was referred to the same clinic at the age of ten years with a scaphocephalic head shape. He had previously undergone vault remodelling at the age of 16 months owing to bicoronal synostosis. At the time of referral, he had an occipito-frontal circumference of 48.5 cm (-2.8 SD), hypertelorism and severe exorbitism. In addition, he had mild maxillary hypoplasia and both 2nd premolars of the lower jaw were absent. Ophthalmic examination showed myopia with divergent strabismus of the right eye associated with reduced visual acuity. A monobloc procedure without distraction (Le Fort III and an advancement of the forehead) was performed.
Due to the severe peri-orbital features a diagnosis of Crouzon syndrome was suggested. Analysis of FGFR2 identified the heterozygous point mutation c.1083A>T [p.(Pro361Pro)]. His father, grandmother and great-grandmother had a similar craniofacial appearance. Based on the pedigree analysis, it is evident that III-1 and III-2 are third cousins and that the FGFR2 mutation present in these two branches is identical by descent (Figure 1C). Apart from III-1 and III-2, none of the other affected family members had undergone craniofacial surgery.
Materials and methods
Ethics approval for the study was obtained from NRES Committee London - Riverside (09/H0706/20) and the Medical Ethical Committee of the Erasmus University Medical Center Rotterdam (MEC-2013-547). Venous blood was collected into PAXgene Blood RNA tubes (Qiagen) from individuals II-1 (Family 1) and III-1 (Family 2), and RNA was extracted according to the associated protocol. cDNA was synthesized using the Fermentas RevertAid First-Strand Synthesis kit with random hexamer primers according to the manufacturer's instructions.
cDNA was amplified using a forward primer in FGFR2 exon IIIa (5′-TCGGAGGAGACGTAGAGTTTGTCTGC-3′) used in combination with a reverse primer in exon 11 (encoding the transmembrane (TM) domain; 5′-TGTTACCTGTCTCCGCAGGGGGATA-3′). DNA bands were cut out and gel purified using the Q-Spin gel extraction kit (Geneflow). Dideoxy sequencing was carried out on the resulting DNA products. The resulting cDNA products were numbered according to NCBI Reference Sequence: NM_000141.4.
The synonymous variants c.1083A>G and c.1083A>T occur at the -2 position of the 5′ (donor) splice site of FGFR2 exon IIIc (Figure 2A). The neural network splice site predictor (http://www.fruitfly.org/seq_tools/splice.html) generates a score for the wild type donor of 0.88, which is reduced to 0.37 by the A>G transition, and to 0.19 by the A>T transversion. In these circumstances, use of a cryptic splice site (score 0.84) within exon IIIc, 51 nucleotides upstream from the end of the exon, is expected based on analysis of a previous mutation c.1084+3A>G . This would lead to an in-frame deletion of 17 amino acids.
Amplification of cDNA from individuals heterozygous for either the FGFR2 c.1083A>G or the c.1083A>T variants demonstrated the presence of two additional bands, not present in the wild type control, at approximately 430 bp and 330 bp (Figure 2B). Sequencing of the normal 479 bp product from these individuals showed complete absence of the mutant allele (illustrated in Figure 2C for II-1 from Family 1), indicating that both mutations abolish use of the normal exon IIIc donor splice site. Sequencing of the ~430 bp product confirmed that the cryptic splice donor within exon IIIc was preferred in the mutant allele (Figure 2D), while the ~330 bp product demonstrated complete skipping of exon IIIc (Figure 2E).
Summary of mutations affecting correct splicing of the FGFR2 exon IIIc donor site
Activation of cryptic splice site
Loss of normal donor site with use of alternative cryptic splice site
Annotated as missense but likely to affect splicing
Everett et al. 1999 
Loss of normal donor site with use of alternative cryptic splice site
Kan et al. 2004 
Loss of normal donor site with use of alternative cryptic splice site
In the cases reported here, the wild type splice donor site is abolished by the A>G or A>T mutations at the -2 position from the intron, leading to the cryptic site being preferred. The apparently greater amount of mutant cDNA products associated with the A>T mutation appears to correlate both with the greater predicted disruption of the splice site and with the more severe phenotype in clinically affected individuals from Family 2 compared with Family 1. However, since the A>G mutation did not support use of the normal exon IIIc donor splice site (Figure 2C), other explanations for these differences are possible, such as differing proportions of cell types in the blood samples analysed, and/or differences in genetic background.
Utilisation of the cryptic donor would lead to an in-frame deletion of the last 17 amino acids of exon IIIc (p.Gly345_Pro361del), including four residues that form specific contacts with the ligand FGF2 . However as craniosynostosis-causing FGFR mutations function in a constitutively active dominant manner , it is also likely that in these individuals a mutant protein is formed which is prone to forming covalently-linked dimers , leading to variable features of Crouzon syndrome.
Our case reports document the range of phenotypic consequences associated with these particular mutations. Whilst a mild crouzonoid phenotype was generally evident, only a minority of individuals developed overt craniosynostosis requiring calvarial surgery. Orthodontic problems may also occur but these were not fully documented in our study. The causes of the clinical variability are unknown, although one potential factor may be the extent of intrauterine fetal head constraint .
In conclusion, mutations near the FGFR2 exon IIIc splice sites should be carefully evaluated as to whether they may be pathogenic, even if they are synonymous or outside the canonical AG/GT splice acceptor/donor sequences. In particular, the mutations described here are associated with variable Crouzon syndrome features and affected families should be counselled as such.
Written informed consent was obtained from the patients for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Patient ascertainment and assessment: JACG, JR, AJMH, SAW, IMJM, AOMW. Experimental analysis: ALF, HL, AMWO. Supervised experiments: TL, AOMW. Drafted paper: ALF, JACG, AOMW. All authors approved the paper.
We thank all families for their participation, J. Frankland and T. Rostron for DNA sequencing, and W. Baggley for clinical photography. This work was funded by the Wellcome Trust (093329 and 102731) and Oxford NIHR Biomedical Research Centre (A.O.M.W).
- Boulet SL, Rasmussen SA, Honein MA: A population-based study of craniosynostosis in metropolitan Atlanta, 1989-2003. Am J Med Genet A. 2008, 146A: 984-991. 10.1002/ajmg.a.32208.View ArticlePubMedGoogle Scholar
- Lajeunie E, Le Merrer M, Bonaiti-Pellie C, Marchac D, Renier D: Genetic study of nonsyndromic coronal craniosynostosis. Am J Med Genet. 1995, 55: 500-504. 10.1002/ajmg.1320550422.View ArticlePubMedGoogle Scholar
- Wilkie AO, Byren JC, Hurst JA, Jayamohan J, Johnson D, Knight SJ, Lester T, Richards PG, Twigg SR, Wall SA: Prevalence and complications of single-gene and chromosomal disorders in craniosynostosis. Pediatrics. 2010, 126: e391-e400. 10.1542/peds.2009-3491.View ArticlePubMedPubMed CentralGoogle Scholar
- Sharma VP, Fenwick AL, Brockop MS, McGowan SJ, Goos JA, Hoogeboom AJ, Brady AF, Jeelani NO, Lynch SA, Mulliken JB, Murray DJ, Phipps JM, Sweeney E, Tomkins SE, Wilson LC, Bennett S, Cornall RJ, Broxholme J, Kanapin A, Johnson D, Wall SA, van der Spek PJ, Mathijssen IMJ, Maxson RE, Twigg SRF, Wilkie AOM: Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet. 2013, 45: 304-307. 10.1038/ng.2531.View ArticlePubMedPubMed CentralGoogle Scholar
- Jabs EW, Li X, Scott AF, Meyers G, Chen W, Eccles M, Mao JI, Charnas LR, Jackson CE, Jaye M: Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet. 1994, 8: 275-279. 10.1038/ng1194-275.View ArticlePubMedGoogle Scholar
- Reardon W, Winter RM, Rutland P, Pulleyn LJ, Jones BM, Malcolm S: Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet. 1994, 8: 98-103. 10.1038/ng0994-98.View ArticlePubMedGoogle Scholar
- Lajeunie E, Ma HW, Bonaventure J, Munnich A, Le Merrer M, Renier D: FGFR2 mutations in Pfeiffer syndrome. Nat Genet. 1995, 9: 108-10.1038/ng0295-108.View ArticlePubMedGoogle Scholar
- Rutland P, Pulleyn LJ, Reardon W, Baraitser M, Hayward R, Jones B, Malcolm S, Winter RM, Oldridge M, Slaney SF, Poole MD, Wilkie AOM: Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nat Genet. 1995, 9: 173-176. 10.1038/ng0295-173.View ArticlePubMedGoogle Scholar
- Schell U, Hehr A, Feldman GJ, Robin NH, Zackai EH, de Die-Smulders C, Viskochil DH, Stewart JM, Wolff G, Ohashi H, Price RA, Cohen MM, Muenke M: Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome. Hum Mol Genet. 1995, 4: 323-328. 10.1093/hmg/4.3.323.View ArticlePubMedGoogle Scholar
- Wilkie AO, Slaney SF, Oldridge M, Poole MD, Ashworth GJ, Hockley AD, Hayward RD, David DJ, Pulleyn LJ, Rutland P, Malcolm S, Winter RM, Reardon W: Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat Genet. 1995, 9: 165-172. 10.1038/ng0295-165.View ArticlePubMedGoogle Scholar
- Przylepa KA, Paznekas W, Zhang M, Golabi M, Bias W, Bamshad MJ, Carey JC, Hall BD, Stevenson R, Orlow S, Cohen MM: Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome. Nat Genet. 1996, 13: 492-494. 10.1038/ng0896-492.View ArticlePubMedGoogle Scholar
- Merrill AE, Sarukhanov A, Krejci P, Idoni B, Camacho N, Estrada KD, Lyons KM, Deixler H, Robinson H, Chitayat D, Curry CJ, Lachman RS, Wilcox WR, Krakow D: Bent bone dysplasia-FGFR2 type, a distinct skeletal disorder, has deficient canonical FGF signaling. Am J Hum Genet. 2012, 90: 550-557. 10.1016/j.ajhg.2012.02.005.View ArticlePubMedPubMed CentralGoogle Scholar
- Agochukwu NB, Solomon BD, Muenke M: Impact of genetics on the diagnosis and clinical management of syndromic craniosynostoses. Childs Nerv Syst. 2012, 28: 1447-1463. 10.1007/s00381-012-1756-2.View ArticlePubMedPubMed CentralGoogle Scholar
- Johnson D, Wilkie AO: Craniosynostosis. Eur J Hum Genet. 2011, 19: 369-376. 10.1038/ejhg.2010.235.View ArticlePubMedPubMed CentralGoogle Scholar
- Mohammadi M, Olsen SK, Ibrahimi OA: Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev. 2005, 16: 107-137. 10.1016/j.cytogfr.2005.01.008.View ArticlePubMedGoogle Scholar
- Eswarakumar VP, Lax I, Schlessinger J: Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 2005, 16: 139-149. 10.1016/j.cytogfr.2005.01.001.View ArticlePubMedGoogle Scholar
- Kan R, Twigg SR, Berg J, Wang L, Jin F, Wilkie AO: Expression analysis of an FGFR2 IIIc 5′ splice site mutation (1084+3A->G). J Med Genet. 2004, 41: e108-10.1136/jmg.2004.018507.View ArticlePubMedPubMed CentralGoogle Scholar
- Podkrajsek KT, Milenkovic T, Odink RJ, der Grinten HL C-v, Bratanic N, Hovnik T, Battelino T: Detection of a complete autoimmune regulator gene deletion and two additional novel mutations in a cohort of patients with atypical phenotypic variants of autoimmune polyglandular syndrome type 1. Eur J Endocrinol. 2008, 159: 633-639. 10.1530/EJE-08-0328.View ArticlePubMedGoogle Scholar
- Twigg SR, Vorgia E, McGowan SJ, Peraki I, Fenwick AL, Sharma VP, Allegra M, Zaragkoulias A, Sadighi Akha E, Knight SJ, Lord H, Lester T, Izatt L, Lampe AK, Mohammed SN, Stewart FJ, Verloes A, Wilson LC, Healy C, Sharpe PT, Hammond P, Hughes J, Taylor S, Johnson D, Wall SA, Mavrothalassitis G, Wilkie AOM: Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet. 2013, 45: 308-313. 10.1038/ng.2539.View ArticlePubMedPubMed CentralGoogle Scholar
- Everett ET, Britto DA, Ward RE, Hartsfield JK: A novel FGFR2 gene mutation in Crouzon syndrome associated with apparent nonpenetrance. Cleft Palate Craniofac J. 1999, 36: 533-541. 10.1597/1545-1569(1999)036<0533:ANFGMI>2.3.CO;2.View ArticlePubMedGoogle Scholar
- Li X, Park WJ, Pyeritz RE, Jabs EW: Effect of splicing of a silent FGFR2 mutation in Crouzon syndrome. Nat Genet. 1995, 9: 232-10.1038/ng0395-232.View ArticlePubMedGoogle Scholar
- Del Gatto F, Breathnach R: A Crouzon syndrome synonymous mutation activates a 5′ splice site within the IIIc exon of the FGFR2 gene. Genomics. 1995, 27 (3): 558-559. 10.1006/geno.1995.1095.View ArticlePubMedGoogle Scholar
- Cornejo-Roldan LR, Roessler E, Muenke M: Analysis of the mutational spectrum of the FGFR2 gene in Pfeiffer syndrome. Hum Genet. 1999, 104: 425-431. 10.1007/s004390050979.View ArticlePubMedGoogle Scholar
- Kress W, Collman H, Busse M, Halliger-Keller B, Mueller CR: Clustering of FGFR2 gene mutations in patients with Pfeiffer and Crouzon syndromes (FGFR2 associated craniosynostosis). Cytogenet Cell Genet. 2000, 91 (1-4): 134-137. 10.1159/000056833.View ArticlePubMedGoogle Scholar
- Plotnikov AN, Hubbard SR, Schlessinger J, Mohammadi M: Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity. Cell. 2000, 101: 413-424. 10.1016/S0092-8674(00)80851-X.View ArticlePubMedGoogle Scholar
- Robertson SC, Meyer AN, Hart KC, Galvin BD, Webster MK, Donoghue DJ: Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain. Proc Natl Acad Sci U S A. 1998, 95: 4567-4572. 10.1073/pnas.95.8.4567.View ArticlePubMedPubMed CentralGoogle Scholar
- Johnson D, Wall SA, Mann S, Wilkie AO: A novel mutation, Ala315Ser, in FGFR2: a gene-environment interaction leading to craniosynostosis?. Eur J Hum Genet. 2000, 8: 571-577. 10.1038/sj.ejhg.5200499.View ArticlePubMedGoogle Scholar
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