http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2014; 27(14): 1502–1506 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2013.860520
CASE STUDY
A familial case of Muenke syndrome. Diverse expressivity of the FGFR3 Pro252Arg mutation – case report and review of the literature Christos Aravidis1, Christopher P. Konialis2, Constantinos G. Pangalos2, and Zoi Kosmaidou3 1
Critical Care Department, Cytogenetics Unit, Evangelismos Hospital, Medical School of Athens University, Athens, Greece, Department of Molecular Genetics and Preimplantation Genetic Diagnosis, Intergenetics Hellas, Diagnostic Genetic Center, Athens, Greece, 3Department of Genetics, Alexandra Hospital, Athens, Greece
2
Abstract
Keywords
Muenke is a fibroblast growth factor receptor 3 (FGFR-3)-associated syndrome, which was first described in late 1990s. Muenke syndrome is an autosomal dominant disorder characterized mainly by coronal suture craniosynostosis, hearing impairment and intellectual disability. The syndrome is defined molecularly by a unique point mutation c.749C4G in exon 7 of the FGFR3 gene which results to an amino acid substitution p.Pro250Arg of the protein product. Despite the fact that the mutation rate at this nucleotide is one of the most frequently described in human genome, few Muenke familial case reports are published in current literature. We describe individuals among three generations of a Greek family who are carriers of the same mutation. Medical record and physical examination of family members present a wide spectrum of clinical manifestations. In particular, a 38-year-old woman and her father appear milder clinical findings regarding craniofacial characteristics compared to her uncle and newborn female child. This familial case illustrates the variable expressivity of Muenke syndrome in association with an identical gene mutation.
Craniosynostosis, fibroblast growth factor receptor 3, Muenke syndrome, Pro250Arg mutation
Introduction Craniosynostosis is characterized by the premature, related to normal brain development, fusion of the skull sutures. This finding could be a clinical sign either as part of a syndrome or as an isolated sporadic case. The prevalence of the disorder is relatively high, with approximately four new cases for every 10 000 live births [1,2] and may occur uni- or bilaterally. Advances in molecular techniques over the past years have led to the identification of some of the key underlying molecular pathways related to this disorder. The most commonly affected genes are fibroblast growth factor receptor genes (FGFRs), muscle segment homeobox 2 gene (MSX2), TWIST1 transcription factor (TWIST1) and fibrillin1 gene (FBN1). Mutations in these genes are correlated with syndromes such as Crouzon, Apert, Pfeiffer, Saethre-Chotzen and Muenke. Craniosynostosis sporadic cases may be related to a de novo mutation in these genes [3], metabolic diseases [1] or could even be associated with increased intrauterine pressure [4]. Additionally, sporadic cases may also result from genetic alterations involving other, currently unidentified, molecular mechanisms.
Address for correspondence: Christos Aravidis, MD, PhD, Clinical Genetics Department, Rudbecklaboratoriet, Akademiska University Hospital, 751 85 Uppsala, Sweden. Tel: +46 73 957 2851. Fax: +46 18 55 40 25. E-mail: [email protected]
History Received 2 May 2013 Accepted 26 October 2013 Published online 28 November 2013
The highest frequency of craniosynostosis syndromatic cases refers to Muenke syndrome, which has a prevalence of 0.8 to 1 per 10 000 live births [3]. Muenke syndrome is characterized by a point mutation in exon 7 of FGFR3 (fibroblast growth factor receptor 3 gene). This mutation leads to various clinical implications such as bicoronal or unicoronal synostosis, macrocephaly, midfacial hypoplasia, thimble shaped middle phalanges, carpal-tarsal fusion, sensorineural hearing loss and developmental delay. The range of these clinical signs varies from absence of findings to more complex clinical manifestations, which is explained by reduced penetrance and diverse expressivity of the mutation [5–7]. The diagnosis of the syndrome is suggested by clinical findings and confirmed by genetic testing for the defining mutation. We present a familial case of Muenke syndrome, after clinical examination, initial diagnosis was held in an adult woman and was confirmed by molecular examination of the p.Pro250Arg mutation. Further investigation revealed additional family members with the mutation (Figure 1).
Clinical report A 38-year-old woman, at 17 weeks of pregnancy, visited the department of Clinical Genetics Alexandra Hospital for prenatal diagnosis and genetic counseling due to advanced age of gestation. Physical examination revealed clinical features pointing towards craniosynostosis.
Familial Muenke syndrome
DOI: 10.3109/14767058.2013.860520
Specifically, she presented distinctive face figures with prominent forehead, facial asymmetry, hypertelorism, downslanting palpebral fissures, long nose, broad philtrum and low adhesion of the auricles with abnormal-shaped helices. No pathological findings were observed in her upper and lower limbs. During the last four years the patient exhibited seizures and received medical treatment with valproic acid, which was ceased with the initiation of pregnancy. The Electroencephalography (EEG) displayed bradyarrhythmia with bilateral distribution, without additional changes in the diagram while she was hyperventilating. Her mental development and intellectual status were normal and she had completed higher education studies at university level. Moreover, she mentioned a hearing loss problem, hence an audiological examination was undergone. The Audiology results reported a bilateral sensorineural hearing loss. The patient’s family report indicated that her father and uncle have similar phenotypic features. In particular, physical examination revealed that proband’s uncle had a more severe clinical image, with an obviously misshapen skull, prominent forehead and downslanting palpebral fissures. No other family
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members exhibited craniofacial dysmorphic image, hearing deficits or neurodevelopmental disabilities. In accordance with the physical examination, clinical diagnosis of craniosynostosis and differential diagnosis of Saethre-Chotzen and Muenke syndrome were set for the case. We provided genetic counseling and the proband decided to continue her pregnancy. We proceeded with amniotic fluid examination for chromosomal aberrations of the fetus. Karyotype displayed no chromosomal aberrations and fetal chromosomes were normal (46, XX). A detailed prenatal ultrasound scan-control during the gestation period revealed a normal fetus with no obvious anatomical abnormalities. Somatometric measurements were always within the normal, for corresponding gestational age, limits. Gestation evolved normally and a girl was delivered at 39 weeks of gestation by normal vaginal delivery with a weight 3630 g (just below the 85th percentile), body length 52 cm (above the 50th percentile) and head circumference 34 cm (above the 50th percentile). Physical examination of the neonate demonstrated the presence of craniosynostosis (Figure 2). Specifically, she exhibited turricephaly, prominent forehead, midfacial hypoplasia, hypertelorism, downslanting palpebral fissures, broad philtrum, micrognathia of the maxilla and low adhesion of the auricles. Infant’s limbs were found normal, therefore there was no indication for further investigation with a hands and feet X-ray test.
Methods and results
Figure 1. Pedigree of family. Members of the family with dysmorphic characteristics are marked black. The proband is indicated by an arrow. Molecular testing was performed for the proband, her child and her father.
We performed genetic test to infant and consecutively to her mother and grandfather to reveal the inheritance pattern. Genomic DNA was isolated from a peripheral blood sample from these individuals and subjected to bi-directional fluorescent automated DNA Sanger sequencing of the coding exon of the TWIST1 gene, exons 7 and 8 of the FGFR2 gene and exon 7 of the FGFR3 gene. Multiplex ligation-dependent probe amplification technique (MLPA) (P054, MRC Holland, Amsterdam, the Netherlands) was also applied for the detection of deletions-duplications of TWIST1 gene. Mutation detection was performed using the Mutation Surveyor software (SoftGenetics, State College, Pennsylvania, USA). Reference sequences: TWIST1:
Figure 2. Phenotypic features of the infant in front (A and B) and side (C) views. A tower shape skull with prominent forehead is presented in both facial profiles. Additional clinical signs are midfacial hypoplasia, hypertelorism, downslanting palpebral fissures (A and B), broad philtrum and micrognathia of the maxilla (B) and low adhesion of the auricles (C).
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NM_000474.3; FGFR2: NM_000141.4; FGFR3: NM_000142.3. The analysis revealed the pathological mutation c.749C4G (p.Pro252Arg) to be present in all examined individuals at a heterozygote state.
Genetic counseling management We recommended that the child should be monitored by a pediatric craniofacial surgeon in regards to her skull development. Corrective surgery for craniosystosis was successfully performed at the age of 11 months. Furthermore, we proposed that a pediatric neurologist and a psychologist should evaluate the child’s mental development for prompt detection of brain anomalies and intellectual disorders. In addition, concerning the child’s hearing development, a referral to an otolaryngologist was provided for an audiological examination. In particular, we suggested an Evoked Otoacoustic Emissions examination (OAE) for an initial evaluation of the child’s hearing status and an additional Auditory Brainstem Response Audiometry test (ABR) at the age of six months for supplementary control. Finally, we advised the mother to perform a prenatal molecular examination for p.Pro250Arg mutation in each subsequent pregnancy, parallel to a detailed ultrasound control.
Discussion More than 180 syndromes have been associated with craniosynostosis as a clinical manifestation [8]. Several cohort studies comprising a wide number of syndromic cases provide substantial evidence that craniosynostosis has a genetic background of a single gene disorder, rather than a more complex chromosomal aberration [9]. The most commonly affected genes that contribute to this specific phenotype are FGFRs, TWIST1, MSX2 and FBN1 and altogether account for more than 80% of all syndromic cases [10]. The underlying molecular pathways, which associate alterations of fibroblast growth factor receptor protein family and craniosynostosis syndromes, are not well understood. The fibroblast growth factor receptors consist of an extracellular ligand domain, a single transmembrane helix domain and an intracellular domain with tyrosine kinase activity. The isoforms of FGFRs vary in their ligand-binding properties and kinase domains; however, they all share a common extracellular region composed of three immunoglobulin (Ig)-like domains (IgI, IgII, IgIII) [11]. FGFRs bind fibroblast growth factors (FGFs) and dimerize in order to affect the downstream intracellular signaling [12,13]. The gene of FGFR3 is located in the short arms of chromosome 4 in chromosomal region 4p16.3, composed of 17 coding exons and has approximately 17 kb length [14]. Mutations of FGFR3 gene are associated with syndromes like achodroplasia, hypochondroplasia, thanatotropic dysplasia type 1, Crouzon with acanthosis nigricans and Muenke syndrome [15,16]. In Muenke syndrome, the responsible mutation is a single transversion (C ! G) in codon 7 of the gene at position 749, which results to a proline-to-arginine amino acid substitution in position 250 of the protein. This specific mutation is a hallmark for the syndrome, it occurs at a rate of 8 106 per haploid genome which is considered one of the highest known
J Matern Fetal Neonatal Med, 2014; 27(14): 1502–1506
mutation rates, for a transversion, in humans [3,6]. The p.Pro252Arg substitution takes place in the conjunctive region between the second and third immunoglobulin-like domains. It is worth noting that similar mutations occur in analogous positions of FGFRs proteins in other syndromes. In particular, in Pfeifer syndrome a p.Pro252Arg change occurs in FGFR1 [17], also a p.Pro253Arg alteration in FGFR2 is present in Pfeifer [18] and Apert syndromes [19]. Structural studies of FGFR protein, with the use of X-ray crystallography, have denoted a closer insight to the molecular mechanism that associates the amino acid conversion in this location to the phenotype. Proline-to-arginine substitutions alter the relative orientation of the Ig-like domains, thus leading to an enhancement of the ligand binding properties [20]. This gain-of-function process of the molecule leads to an amplification of the osteoblastic activity, including increased proliferation, differentiation and apoptosis of the osteoblasts bordering the cranial sutures, thus resulting to craniosynostosis phenomena [21,22]. The normal fusion of cranial sutures is completed approximately at the 18th month of age. When a suture is prematurely closed, the cranial bone development stops along this margin. Excessive bone growth at other sutures sites leads to skull distortion. The simultaneous fusion of both coronal sutures (bilateral) induces symmetrical brachycephaly or turribrachycephaly (tower-shaped skull) and midface hypoplasia. Infant’s facial figures are accordant to the abovementioned state (Figure 2). The involvement of other sutures may result in different kinds of skull shapes such as scaphocephaly, plagiocephaly, trigonocephaly or even a cloverleaf skull. The proband reported bilateral hearing impairment. This is a common finding of individuals with Muenke syndrome and refers mainly to sensorineural hearing loss at low frequencies. Several researchers support that at least one-third of Muenke syndrome cases exhibit sensorineural hearing loss [15,23], while others suggest that all affected individuals have a degree of hearing problems [24–26]. A plausible explanation for the wide range of outcomes from the above-mentioned studies may be the usage of different diagnostic approaches and methods, and the reality is more likely to the latter suggestion. FGFR3 is expressed in developing pillar, Deiters’ cells and developing outer hair cells of the inner ear, thus playing a significant key role in cell differentiation and homeostasis [27,28]. The p.Pro252Arg mutation and the subsequent over-activation of FGF signaling affect the normal cochlear sensory development and lead to hearing impairment [24,29,30]. Additional common findings in patients with Muenke syndrome are developmental delay, intellectual disability and learning disorders [15]. The possible underlying mechanism could be related either to hearing loss or to the direct impact of craniosynostosis on normal brain development. Hearing impairment could lead to learning disorder and developmental delay in proportion to the extent of hearing loss and therefore a detailed audiological examination is always suggested in such cases [31]. Alternatively, craniosynostosis could increase intracranial pressure (ICP) or directly affect the normal brain development especially the medial temporal structures, thus resulting to dysgenesis of those regions and intellectual
Familial Muenke syndrome
DOI: 10.3109/14767058.2013.860520
disability [31,32]. It should also be noted that FGFR3 is found to be expressed at high levels during brain embryogenesis [33,34] with a significant role in the hippocampus [35]. Hence, an alternate pathway to developmental delay may be related to the deregulation of FGFR3 in individuals with Muenke syndrome. Only a few references in the literature point out seizures as a clinical manifestation of Muenke syndrome [36]. Epileptic seizures are associated with dysgenesis of medial temporal lobe structures and other brain regions such as the corpus callosum, the Ammon’s horn and the hippocampus [32,37,38]. In our case, we have no real evidence for the correlation between the mother’s seizures and a specific brain anatomical disorder since she has never performed a brain imaging scan. Finally, it is worth noting that Muenke syndrome has a diversity regarding the penetrance and expressivity of the p.Pro252Arg mutation in FGFR3. This clinical diversity among family members is also observed in our case. The mother and her father have milder symptoms compared to the child and uncle. In addition, it is described that females usually present more severe clinical signs compared to males [24,39], which is observed in the described infant’s phenotype. Moreover, in most familial cases there is a distinct paternal origin and a correlation to increased paternal age [6,39]. In this case, the mutation is originated from the infant’s grandfather, although no further information regarding before him generations were available. In summary, the heterogeneity of the syndrome results in a wide spectrum of clinical manifestations, which perplex diagnosis. Specifically, the identification of affected members within a family is a complex procedure and selection of individuals to be tested for the mutation should be attentive. Reporting familial cases of Muenke syndrome is a helpful approach to understand the clinical diversity of this disorder and can provide physicians with elements and clues in order to reach the correct diagnosis, prompt molecular control and offer genetic consultation.
5.
6. 7. 8. 9. 10. 11. 12. 13.
14.
15.
16. 17. 18. 19. 20. 21.
Acknowledgements We would like to thank the family members participating in this study for their kind cooperation and their permission to publish the results.
22. 23.
Declaration of interest The authors declare no conflict of interest.
24.
References 1. Cohen Jr MM, MacLean RE, eds. Anatomic, genetic, nosologic, diagnostic, and psychosocial considerations. In: Craniosynostosis. Diagnosis, evaluation, and management. 2nd ed. New York: Oxford University Press; 2000:119–43. 2. Boulet SL, Rasmussen SA, Honein MA. A population-based study of craniosynostosis in metropolitan Atlanta, 1989–2003. Am J Med Genet Part A 2008;146:984–91. 3. Moloney DM, Wall SA, Ashworth GJ, et al. Prevalence of Pro250Arg mutation of fibroblast growth factor receptor 3 in coronal craniosynostosis. Lancet 1997;349:1059–62. 4. Koskinen-Moffett L, Moffett BC. Sutures and intrauterine deformation. In: Persing JA, Edgerton MT, Jane JA, eds. Scientific
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foundation and surgical treatment of craniosynostosis. Baltimore: Williams & Wilkins; 1989:96–106. Graham JM, Braddock SR, Mortier GR, et al. Syndrome of coronal craniosynostosis with brachydactyl and carpal/tarsal coalition due to Pro250Arg mutation in FGFR3 gene. Am J Med Genet 1998;77: 322–9. Rannan-Eliya SV, Taylor IB, de Heer IM, et al. Paternal origin of FGFR3 mutations in Muenke-type craniosynostosis. Hum Genet 2004;115:200–7. Escobar LF, Hiett AK, Marnocha A. Significant phenotypic variability of Muenke syndrome in identical twins. Am J Med Genet 2009;149A:1273–6. Kimonis V, Gold JA, Hoffman TL, et al. Genetics of craniosynostosis. Semin Pediatr Neurol 2007;14:150–61. Wilkie AOM, Byren JC, Hurst JA, et al. Prevalence and complications of single gene and chromosomal disorders in craniosynostosis. Pediatrics 2010;126:391–400. Johnson D, Wilkie A. Craniosynostosis. Eur J Hum Genet 2011;19: 369–76. Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol 2001;2:REVIEWS 3005.1–3005.12. Coutts J, Gallagher J. Receptors for fibroblast growth factors. Immunol Cell Biol 1995;73:584–9. Ibrahimi O, Zhang F, Eliseenkova A, et al. Proline to arginine mutations in FGF receptors 1 and 3 result in Pfeiffer and Muenke craniosynostosis syndromes through enhancement of FGF binding affinity. Hum Molec Genet 2004;13:69–78. Perez-Castro AV, Wilson J, Altherr MR. Genomic organization of the human fibroblast growth factor receptor 3 (FGFR3) gene and comparative sequence analysis with the mouse Fgfr3 gene. Genomics 1997;41:10–16. Muenke M, Gripp KW, McDonald-McGinn DM, et al. A unique point mutation in the fibroblast growth factor receptor 3 gene (FGFR3) defines a new craniosynostosis syndrome. Am J Hum Genet 1997;60:555–64. Aleck K. Craniosynostosis syndromes in the genomic era. Semin Pediatr Neurol 2009;11:256–61. Schell U, Hehr A, Feldman GJ, et al. Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome. Hum Mol Genet 1995;4:323–8. Muenke M, Schell U, Hehr A, et al. A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nature Genet 1994;8:269–74. Wilkie AOM. Craniosynostosis: genes and mechanisms. Hum Mol Genet 1997;6:1647–56. Cunningham ML, Seto ML, Ratisoontorn C, et al. Syndromic craniosynostosis: from history to hydrogen bonds. Orthod Craniofac Res 2007;10:67–81. Iseki S, Wilkie AOM, Morriss-Kay GM. Fgfr1 and Fgfr2 have distinct differentiation and proliferation-related roles in the developing mouse skull vault. Development 1999;126: 5611–20. Holmes G, Rothschild G, Roy UB, et al. Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology. Dev Biol 2009;328:273–84. de Jong T, Bannink N, Bredero-Boelhouwer HH, et al. Long-term functional outcome in 167 patients with syndromic craniosynostosis; defining a syndrome-specific risk profile. J Plast Reconstr Aesthet Surg 2010;63:1635–41. Doherty ES, Lacbawan F, Hadley DW, et al. Muenke syndrome (FGFR3-related craniosynostosis): expansion of the phenotype and review of the literature. Am J Med Genet 2007; 143A:3204–15. Honnebier MB, Cabiling DS, Hetlinger M, et al. The natural history of patients treated for FGFR3-associated (Muenke-type) craniosynostosis. Plast Reconstr Surg 2008;121:919–31. Mansour SL, Twigg SRF, Freeland RM, et al. Hearing loss in a mouse model of Muenke syndrome. Hum Mol Genet 2009;18: 43–50. Colvin, JS, Bohne BA, Harding GW, et al. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 1996;12:390–7. Hayashi T, Cunningham D, Bermingham-McDonogh O. Loss of FGFR3 leads to excess hair cell development in the mouse organ of corti. Dev Dyn 2007;236:525–33.
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29. Wright TJ, Mansour SL. FGF signaling in ear development and innervation. Curr Top Dev Biol 2003;57:225–9. 30. Schimmang T. Expression and functions of FGF ligands during early otic development. Int J Dev Biol 2007;51:473–81. 31. Agochukwu NB, Doherty ES, Muenke M. Muenke syndrome. In: Pagon RA, Bird TD, Dolan CR, et al., eds. GeneReviewsÔ [Internet]. Seattle: University of Washington; 2010. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1415/ [last accessed 7 Dec 2010]. 32. Grosso S, Farnetani A, Berardi R, et al. Medial temporal lobe dysgenesis in Muenke syndrome and hypochondroplasia. Am J Med Genet 2003;120A:88–91. 33. Peters KG, Ornitz DM, Werner S, Williams LT. Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol 1993;155:423–30. 34. Reimers D, Lopez-Toledano MA, Mason I. Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell
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progeny. Modulation of neuronal and glial lineages by basic FGF treatment. Neurol Res 2001;23:612–21. Lu L, Airey DC, Williams RW. Complex trait analysis of the hippocampus: mapping and biometric analysis of two nove loci with specific effects on hippocampal structure in mice. J Neurosci 2001;15:3503–14. Agochukwu NB, Solomon BD, Gropman AL, Muenke M. Epilepsy in Muenke syndrome: FGFR3-related craniosynostosis. Pediatr Neurol 2012;47:355–61. Sabatino G, Di Rocco F, Zampino G, et al. Muenke syndrome. Childs Nerv Syst 2004;20:297–301. Ridgway EB, Wu JK, Sullivan SR, et al. Craniofacial growth in patients with FGFR3 Pro250Arg mutation after fronto-orbital advancement in infancy. J Craniofac Surg 2011;22:455–61. Lajeunie E, Ghouzzi VE, Le Merrer M, et al. Sex related expressivity of the phenotype in coronal craniosynostosis caused by the recurrent P250R FGFR3 mutation. J Med Genet 1999;6:9–13.
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CASE STUDY
A familial case of Muenke syndrome. Diverse expressivity of the FGFR3 Pro252Arg mutation – case report and review of the literature Christos Aravidis1, Christopher P. Konialis2, Constantinos G. Pangalos2, and Zoi Kosmaidou3 1
Critical Care Department, Cytogenetics Unit, Evangelismos Hospital, Medical School of Athens University, Athens, Greece, Department of Molecular Genetics and Preimplantation Genetic Diagnosis, Intergenetics Hellas, Diagnostic Genetic Center, Athens, Greece, 3Department of Genetics, Alexandra Hospital, Athens, Greece
2
Abstract
Keywords
Muenke is a fibroblast growth factor receptor 3 (FGFR-3)-associated syndrome, which was first described in late 1990s. Muenke syndrome is an autosomal dominant disorder characterized mainly by coronal suture craniosynostosis, hearing impairment and intellectual disability. The syndrome is defined molecularly by a unique point mutation c.749C4G in exon 7 of the FGFR3 gene which results to an amino acid substitution p.Pro250Arg of the protein product. Despite the fact that the mutation rate at this nucleotide is one of the most frequently described in human genome, few Muenke familial case reports are published in current literature. We describe individuals among three generations of a Greek family who are carriers of the same mutation. Medical record and physical examination of family members present a wide spectrum of clinical manifestations. In particular, a 38-year-old woman and her father appear milder clinical findings regarding craniofacial characteristics compared to her uncle and newborn female child. This familial case illustrates the variable expressivity of Muenke syndrome in association with an identical gene mutation.
Craniosynostosis, fibroblast growth factor receptor 3, Muenke syndrome, Pro250Arg mutation
Introduction Craniosynostosis is characterized by the premature, related to normal brain development, fusion of the skull sutures. This finding could be a clinical sign either as part of a syndrome or as an isolated sporadic case. The prevalence of the disorder is relatively high, with approximately four new cases for every 10 000 live births [1,2] and may occur uni- or bilaterally. Advances in molecular techniques over the past years have led to the identification of some of the key underlying molecular pathways related to this disorder. The most commonly affected genes are fibroblast growth factor receptor genes (FGFRs), muscle segment homeobox 2 gene (MSX2), TWIST1 transcription factor (TWIST1) and fibrillin1 gene (FBN1). Mutations in these genes are correlated with syndromes such as Crouzon, Apert, Pfeiffer, Saethre-Chotzen and Muenke. Craniosynostosis sporadic cases may be related to a de novo mutation in these genes [3], metabolic diseases [1] or could even be associated with increased intrauterine pressure [4]. Additionally, sporadic cases may also result from genetic alterations involving other, currently unidentified, molecular mechanisms.
Address for correspondence: Christos Aravidis, MD, PhD, Clinical Genetics Department, Rudbecklaboratoriet, Akademiska University Hospital, 751 85 Uppsala, Sweden. Tel: +46 73 957 2851. Fax: +46 18 55 40 25. E-mail: [email protected]
History Received 2 May 2013 Accepted 26 October 2013 Published online 28 November 2013
The highest frequency of craniosynostosis syndromatic cases refers to Muenke syndrome, which has a prevalence of 0.8 to 1 per 10 000 live births [3]. Muenke syndrome is characterized by a point mutation in exon 7 of FGFR3 (fibroblast growth factor receptor 3 gene). This mutation leads to various clinical implications such as bicoronal or unicoronal synostosis, macrocephaly, midfacial hypoplasia, thimble shaped middle phalanges, carpal-tarsal fusion, sensorineural hearing loss and developmental delay. The range of these clinical signs varies from absence of findings to more complex clinical manifestations, which is explained by reduced penetrance and diverse expressivity of the mutation [5–7]. The diagnosis of the syndrome is suggested by clinical findings and confirmed by genetic testing for the defining mutation. We present a familial case of Muenke syndrome, after clinical examination, initial diagnosis was held in an adult woman and was confirmed by molecular examination of the p.Pro250Arg mutation. Further investigation revealed additional family members with the mutation (Figure 1).
Clinical report A 38-year-old woman, at 17 weeks of pregnancy, visited the department of Clinical Genetics Alexandra Hospital for prenatal diagnosis and genetic counseling due to advanced age of gestation. Physical examination revealed clinical features pointing towards craniosynostosis.
Familial Muenke syndrome
DOI: 10.3109/14767058.2013.860520
Specifically, she presented distinctive face figures with prominent forehead, facial asymmetry, hypertelorism, downslanting palpebral fissures, long nose, broad philtrum and low adhesion of the auricles with abnormal-shaped helices. No pathological findings were observed in her upper and lower limbs. During the last four years the patient exhibited seizures and received medical treatment with valproic acid, which was ceased with the initiation of pregnancy. The Electroencephalography (EEG) displayed bradyarrhythmia with bilateral distribution, without additional changes in the diagram while she was hyperventilating. Her mental development and intellectual status were normal and she had completed higher education studies at university level. Moreover, she mentioned a hearing loss problem, hence an audiological examination was undergone. The Audiology results reported a bilateral sensorineural hearing loss. The patient’s family report indicated that her father and uncle have similar phenotypic features. In particular, physical examination revealed that proband’s uncle had a more severe clinical image, with an obviously misshapen skull, prominent forehead and downslanting palpebral fissures. No other family
1503
members exhibited craniofacial dysmorphic image, hearing deficits or neurodevelopmental disabilities. In accordance with the physical examination, clinical diagnosis of craniosynostosis and differential diagnosis of Saethre-Chotzen and Muenke syndrome were set for the case. We provided genetic counseling and the proband decided to continue her pregnancy. We proceeded with amniotic fluid examination for chromosomal aberrations of the fetus. Karyotype displayed no chromosomal aberrations and fetal chromosomes were normal (46, XX). A detailed prenatal ultrasound scan-control during the gestation period revealed a normal fetus with no obvious anatomical abnormalities. Somatometric measurements were always within the normal, for corresponding gestational age, limits. Gestation evolved normally and a girl was delivered at 39 weeks of gestation by normal vaginal delivery with a weight 3630 g (just below the 85th percentile), body length 52 cm (above the 50th percentile) and head circumference 34 cm (above the 50th percentile). Physical examination of the neonate demonstrated the presence of craniosynostosis (Figure 2). Specifically, she exhibited turricephaly, prominent forehead, midfacial hypoplasia, hypertelorism, downslanting palpebral fissures, broad philtrum, micrognathia of the maxilla and low adhesion of the auricles. Infant’s limbs were found normal, therefore there was no indication for further investigation with a hands and feet X-ray test.
Methods and results
Figure 1. Pedigree of family. Members of the family with dysmorphic characteristics are marked black. The proband is indicated by an arrow. Molecular testing was performed for the proband, her child and her father.
We performed genetic test to infant and consecutively to her mother and grandfather to reveal the inheritance pattern. Genomic DNA was isolated from a peripheral blood sample from these individuals and subjected to bi-directional fluorescent automated DNA Sanger sequencing of the coding exon of the TWIST1 gene, exons 7 and 8 of the FGFR2 gene and exon 7 of the FGFR3 gene. Multiplex ligation-dependent probe amplification technique (MLPA) (P054, MRC Holland, Amsterdam, the Netherlands) was also applied for the detection of deletions-duplications of TWIST1 gene. Mutation detection was performed using the Mutation Surveyor software (SoftGenetics, State College, Pennsylvania, USA). Reference sequences: TWIST1:
Figure 2. Phenotypic features of the infant in front (A and B) and side (C) views. A tower shape skull with prominent forehead is presented in both facial profiles. Additional clinical signs are midfacial hypoplasia, hypertelorism, downslanting palpebral fissures (A and B), broad philtrum and micrognathia of the maxilla (B) and low adhesion of the auricles (C).
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NM_000474.3; FGFR2: NM_000141.4; FGFR3: NM_000142.3. The analysis revealed the pathological mutation c.749C4G (p.Pro252Arg) to be present in all examined individuals at a heterozygote state.
Genetic counseling management We recommended that the child should be monitored by a pediatric craniofacial surgeon in regards to her skull development. Corrective surgery for craniosystosis was successfully performed at the age of 11 months. Furthermore, we proposed that a pediatric neurologist and a psychologist should evaluate the child’s mental development for prompt detection of brain anomalies and intellectual disorders. In addition, concerning the child’s hearing development, a referral to an otolaryngologist was provided for an audiological examination. In particular, we suggested an Evoked Otoacoustic Emissions examination (OAE) for an initial evaluation of the child’s hearing status and an additional Auditory Brainstem Response Audiometry test (ABR) at the age of six months for supplementary control. Finally, we advised the mother to perform a prenatal molecular examination for p.Pro250Arg mutation in each subsequent pregnancy, parallel to a detailed ultrasound control.
Discussion More than 180 syndromes have been associated with craniosynostosis as a clinical manifestation [8]. Several cohort studies comprising a wide number of syndromic cases provide substantial evidence that craniosynostosis has a genetic background of a single gene disorder, rather than a more complex chromosomal aberration [9]. The most commonly affected genes that contribute to this specific phenotype are FGFRs, TWIST1, MSX2 and FBN1 and altogether account for more than 80% of all syndromic cases [10]. The underlying molecular pathways, which associate alterations of fibroblast growth factor receptor protein family and craniosynostosis syndromes, are not well understood. The fibroblast growth factor receptors consist of an extracellular ligand domain, a single transmembrane helix domain and an intracellular domain with tyrosine kinase activity. The isoforms of FGFRs vary in their ligand-binding properties and kinase domains; however, they all share a common extracellular region composed of three immunoglobulin (Ig)-like domains (IgI, IgII, IgIII) [11]. FGFRs bind fibroblast growth factors (FGFs) and dimerize in order to affect the downstream intracellular signaling [12,13]. The gene of FGFR3 is located in the short arms of chromosome 4 in chromosomal region 4p16.3, composed of 17 coding exons and has approximately 17 kb length [14]. Mutations of FGFR3 gene are associated with syndromes like achodroplasia, hypochondroplasia, thanatotropic dysplasia type 1, Crouzon with acanthosis nigricans and Muenke syndrome [15,16]. In Muenke syndrome, the responsible mutation is a single transversion (C ! G) in codon 7 of the gene at position 749, which results to a proline-to-arginine amino acid substitution in position 250 of the protein. This specific mutation is a hallmark for the syndrome, it occurs at a rate of 8 106 per haploid genome which is considered one of the highest known
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mutation rates, for a transversion, in humans [3,6]. The p.Pro252Arg substitution takes place in the conjunctive region between the second and third immunoglobulin-like domains. It is worth noting that similar mutations occur in analogous positions of FGFRs proteins in other syndromes. In particular, in Pfeifer syndrome a p.Pro252Arg change occurs in FGFR1 [17], also a p.Pro253Arg alteration in FGFR2 is present in Pfeifer [18] and Apert syndromes [19]. Structural studies of FGFR protein, with the use of X-ray crystallography, have denoted a closer insight to the molecular mechanism that associates the amino acid conversion in this location to the phenotype. Proline-to-arginine substitutions alter the relative orientation of the Ig-like domains, thus leading to an enhancement of the ligand binding properties [20]. This gain-of-function process of the molecule leads to an amplification of the osteoblastic activity, including increased proliferation, differentiation and apoptosis of the osteoblasts bordering the cranial sutures, thus resulting to craniosynostosis phenomena [21,22]. The normal fusion of cranial sutures is completed approximately at the 18th month of age. When a suture is prematurely closed, the cranial bone development stops along this margin. Excessive bone growth at other sutures sites leads to skull distortion. The simultaneous fusion of both coronal sutures (bilateral) induces symmetrical brachycephaly or turribrachycephaly (tower-shaped skull) and midface hypoplasia. Infant’s facial figures are accordant to the abovementioned state (Figure 2). The involvement of other sutures may result in different kinds of skull shapes such as scaphocephaly, plagiocephaly, trigonocephaly or even a cloverleaf skull. The proband reported bilateral hearing impairment. This is a common finding of individuals with Muenke syndrome and refers mainly to sensorineural hearing loss at low frequencies. Several researchers support that at least one-third of Muenke syndrome cases exhibit sensorineural hearing loss [15,23], while others suggest that all affected individuals have a degree of hearing problems [24–26]. A plausible explanation for the wide range of outcomes from the above-mentioned studies may be the usage of different diagnostic approaches and methods, and the reality is more likely to the latter suggestion. FGFR3 is expressed in developing pillar, Deiters’ cells and developing outer hair cells of the inner ear, thus playing a significant key role in cell differentiation and homeostasis [27,28]. The p.Pro252Arg mutation and the subsequent over-activation of FGF signaling affect the normal cochlear sensory development and lead to hearing impairment [24,29,30]. Additional common findings in patients with Muenke syndrome are developmental delay, intellectual disability and learning disorders [15]. The possible underlying mechanism could be related either to hearing loss or to the direct impact of craniosynostosis on normal brain development. Hearing impairment could lead to learning disorder and developmental delay in proportion to the extent of hearing loss and therefore a detailed audiological examination is always suggested in such cases [31]. Alternatively, craniosynostosis could increase intracranial pressure (ICP) or directly affect the normal brain development especially the medial temporal structures, thus resulting to dysgenesis of those regions and intellectual
Familial Muenke syndrome
DOI: 10.3109/14767058.2013.860520
disability [31,32]. It should also be noted that FGFR3 is found to be expressed at high levels during brain embryogenesis [33,34] with a significant role in the hippocampus [35]. Hence, an alternate pathway to developmental delay may be related to the deregulation of FGFR3 in individuals with Muenke syndrome. Only a few references in the literature point out seizures as a clinical manifestation of Muenke syndrome [36]. Epileptic seizures are associated with dysgenesis of medial temporal lobe structures and other brain regions such as the corpus callosum, the Ammon’s horn and the hippocampus [32,37,38]. In our case, we have no real evidence for the correlation between the mother’s seizures and a specific brain anatomical disorder since she has never performed a brain imaging scan. Finally, it is worth noting that Muenke syndrome has a diversity regarding the penetrance and expressivity of the p.Pro252Arg mutation in FGFR3. This clinical diversity among family members is also observed in our case. The mother and her father have milder symptoms compared to the child and uncle. In addition, it is described that females usually present more severe clinical signs compared to males [24,39], which is observed in the described infant’s phenotype. Moreover, in most familial cases there is a distinct paternal origin and a correlation to increased paternal age [6,39]. In this case, the mutation is originated from the infant’s grandfather, although no further information regarding before him generations were available. In summary, the heterogeneity of the syndrome results in a wide spectrum of clinical manifestations, which perplex diagnosis. Specifically, the identification of affected members within a family is a complex procedure and selection of individuals to be tested for the mutation should be attentive. Reporting familial cases of Muenke syndrome is a helpful approach to understand the clinical diversity of this disorder and can provide physicians with elements and clues in order to reach the correct diagnosis, prompt molecular control and offer genetic consultation.
5.
6. 7. 8. 9. 10. 11. 12. 13.
14.
15.
16. 17. 18. 19. 20. 21.
Acknowledgements We would like to thank the family members participating in this study for their kind cooperation and their permission to publish the results.
22. 23.
Declaration of interest The authors declare no conflict of interest.
24.
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