7444 JMed Genet 1996;33:744-748 A recurrent mutation, ala39 1 glu, in the transmembrane region of FGFR3 causes Crouzon J Med Genet: first published as 10.1136/jmg.33.9.744 on 1 September 1996. Downloaded from syndrome and acanthosis nigricans D Wilkes, P Rutland, L J Pulleyn, W Reardon, C Moss, J P Ellis, R M Winter, S Malcolm Abstract and Jackson-Weiss syndromes as an example of Mutations in the fibroblast growth factor the same phenomenon. receptor 2 (FGFR2) gene have previously Crouzon syndrome, which is clinically differ- been identified in Crouzon syndrome, an entiated from these other syndromic forms of autosomal dominant condition involving craniosynostosis by the degree of proptosis, premature fusion of the cranial sutures. maxillary hypoplasia, and the absence of limb Several different missense and other mu- malformations, has now been associated with tations have been identified in Crouzon over 30 mutations at the FGFR2 locus, syndrome patients, clustering around the missense, splice site, and small in frame third immunoglobulin-like domain. We deletions.4 6 13-15 Mutations are located in both report here the identification of a muta- the first and second half of the third tion in the transmembrane region of immunoglobulin-like domain, and the non- FGFR3, common to three unrelated pa- random pattern of mutations has been dis- tients with classical Crouzon syndrome cussed elsewhere.12 Although the range of and acanthosis nigricans, a dermatologi- FGFR2 mutations associated with Crouzon cal condition associated with thickening syndrome has been broad, the original linkage and abnormal pigmentation of the skin. data showed no evidence of heterogeneity.6 17 The mutation within the FGFR3 tran- To date, analysis of the FGFR2 locus has iden- script was determined by direct sequenc- tified mutations in approximately 66% of our ing as a specific gcg to gag transversion, Crouzon syndrome cohort of 70 patients, none resulting in an amino acid substitution of whom had acanthosis nigricans. ala391glu within the transmembrane re- One of the less common associations seen in gion. a subgroup ofpatients with Crouzon syndrome (37Med Genet 1996;33:744-748) is acanthosis nigricans. This velvety hyperpla- sia of the skin, with accentuation of skin mark- Keywords: Crouzon syndrome; acanthosis nigricans; ings and hyperpigmentation, is usually found http://jmg.bmj.com/ fibroblast growth factor receptor 3 (FGFR3). on the neck, axillae, and other flexural regions of the body, but acanthosis nigricans is, in gen- Mothercare Unit of eral, recognised to be clinically heterogeneous. Clinical Genetics and Fetal Medicine and Several clinically distinct monogenic disorders Although uncommon, the association with Molecular Genetics present with craniosynostosis, the premature Crouzon syndrome is clear, 11 such cases hav- of Child Unit, Institute fusion of the skull bone sutures.1 2 Initially ing been identified by Koizumi et al.'8 These Health, 30 Guilford on September 23, 2021 by guest. Protected copyright. Street, London WClN delineated purely on clinical grounds, several authors drew attention to some atypical lEH,UK reports over the last year have identified the features of the Crouzon associated acanthosis D Wilkes mutational basis of the phenotypes and posed nigricans, specifically the early onset of the P Rutland of L J Pulleyn new questions as to the mechanisms by which pigmentation and the unusual distribution W Reardon the resultant malformations arise.3 Initially the affected skin, particularly involving the R M Winter described in Crouzon syndrome, mutations at perioral region. S Malcolm the fibroblast growth factor receptor 2 Mutations within FGFR3 have been identi- Department of (FGFR2) locus on chromosome 10 have since fied in discrete skeletal dysplasias. Four such Dermatology, been identified in Apert and Jackson-Weiss conditions have now been causatively associ- Birmingham syndromes.46 ated with FGFR3 mutations: achondroplasia, Children's Hospital, in Ladywood, A mutation in the related gene on chromo- in which virtually all mutations are the Birmingham B16 8ET, some 8, FGFR1, has been characterised in transmembrane region of the receptor,9-2' UK Pfeiffer syndrome,7 but locus heterogeneity in hypochondroplasia, in which there are two C Moss this syndrome was established with the obser- mutations in the tyrosine kinase domain,22 23 Department of vation of Pfeiffer syndrome cases having muta- and thanatophoric dysplasia types I and II Dermatology, Princess tions at the FGFR2 locus.'-" More than three (TDI and II). TDI, in which the femora are Margaret Hospital, such mutations, all resulting in the substitution curved, has been described in association with Okus Road, Swindon, a Wilts SN1 4JU,UK of cys342, have been identified in both missense mutations involving creation of J P Ellis Crouzon and Pfeiffer syndrome cases and the cysteine in the linker region between factors governing the development of these immunoglobulin-like domains 2 and 324 25 and Correspondence to: Dr Reardon. clinically distinct malformation syndromes, read through mutations of the stop codon.26 arising from the same initial mutation, remain Type II, characterised by relatively long and Received 4 March 1996 et have cited an identical straight femora, has been described in associ- Revised version accepted for unclear."2 Gorry all' publication 28 May 1996 mutation (ala344gly) in FGFR2 in Crouzon ation with a tyrosine kinase domain mutation A recurrent mutation, ala391glu, causes Crouzon syndrome and acanthosis nigricans 745 B J Med Genet: first published as 10.1136/jmg.33.9.744 on 1 September 1996. Downloaded from .. 1: .i. I 1: .i. .: I..k Figure 1 Postoperative appearance of case 1. Note the mildfacial asymmetry and perioral acanthosis nigricans (A) and theflexural skin thickening and acanthosis nigricans (B). (All photographs reproduced with permission.) (K650E) of FGFR3."4 As both types may be and other clinical parameters were normal. associated with the severe skull malformation, Proptosis and skull malformation were evident clover leaf skull, TDI and II represent evidence from birth and craniosynostosis was confirmed that FGFR3 mutations may also have cranio- radiologically. Motor and intellectual develop- synostosis clinically. We now extend the associ- ment have been normal and the craniosynosto- ation between FGFR3 mutations and cranio- sis has been managed surgically. At the age of synostosis by reporting a single specific 14 years, he was referred to a dermatologist mutation, ala39 1 glu, in the transmembrane (CM) with a history of dry skin on the face region of FGFR3 in three unrelated patients since the age of 9. Clinical examination showed with Crouzon syndrome and acanthosis nigri- extensive acanthosis nigricans of the axilla, http://jmg.bmj.com/ cans. This is only 11 amino acids away, within neck, and, to a lesser extent, the groin. The the transmembrane domain, from the pre- periorbital and perioral skin was prominently dominant mutation causing achondroplasia. affected but there was no palmoplantar or mucosal involvement. There was generalised Patients and methods skin thickening, particularly over the trunk. In CASE 1 addition several small, warty acanthomas were This male patient (fig 1) was born to clinically noted, as were several melanocytic naevi on the on September 23, 2021 by guest. Protected copyright. normal parents following a normal pregnancy face and upper trunk. and delivery. Birth weight, head circumference, CASE 2 This female patient was born to clinically nor- mal parents (fig 2). The early clinical course was dominated by problems with feeding and failure to thrive. Incomplete choanal atresia led to associated respiratory difficulties. Her head circumference was on the 50th centile, dispro- portionately large for her height and weight, which were both below the 3rd centile. There was proptosis clinically and skull x ray confirmed premature bicoronal synostosis at the age of 8 months. There was midface hypo- plasia and surgical correction of the craniofa- cial malformations was performed at the age of 10 months. Motor and intellectual develop- ment were normal. Reviewed dermatologically at the age of 6 years (JPE), there was a notable degree of thickening of the skin around the neck and fossae. Velvety thickening and hyper- pigmentation, characteristic of acanthosis nig- ricans, were seen on the neck, axillae, antecu- 746 Wilkes, Rutland, Pulleyn, Reardon, Moss, Ellis, Winter, Malcolm logical involvement was striking. There were no other abnormalities and a recent blood sugar evaluation was normal. J Med Genet: first published as 10.1136/jmg.33.9.744 on 1 September 1996. Downloaded from PCR ANALYSIS DNA was extracted from lymphocytes by salt- ing out.27 Primers amplifying the entire trans- membrane domain of FGFR3 from genomic DNA were used as previously published.9 A 164 bp product results. The primer sequences 5'AGGAGCT- GGTGGAGGCTGA-3' and 5'BioGGAG- ATCTTGTGCACGGTGG-3' were designed to amplify the entire transmembrane domain of FGFR3 from genomic DNA. PCR condi- tions were 200 ng of genomic DNA, 10 mmol.l' Tris, pH 8.3, 1.5 mmol.l1 MgCl, 50 mmol.l' KCI, 200 gmol.l` dNTP, 0.7 !mol l-l c32P-dCTP, 50 pmol of each primer, and 0.5 U Taq polymerase in a final volume of 50 gl: 30 cycles of 94'C x 30 seconds, 64'C x 30 seconds, 72°C x 40 seconds. RESTRICTION DIGESTION PCR products were purified by phenol/ chloroform ethanol precipitation and restric- Figure 3 Lateral view of case 3 showing brachycephaly tion digestion was performed according to the and maxillary hypoplasia. Note the warty acanthomas over manufacturer's specifications (New England the lower neck (A) and severe acanthosis nigricans over the Biolabs). Resolution of fragment products was neck (B). performed on a 3% agarose gel. bital fossae, and around the umbilicus and the DNA SEQUENCING angles of the mouth. PCR products were sequenced directly by including a single 5' biotinylated primer in the CASE 3 PCR reaction. PCR products were then This 61 year old man was referred for genetic captured and immobilised to Dynabeads counselling for Crouzon syndrome (fig 3).