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Novel BRAF mutation in a patient with LEOPARD syndrome and normal intelligence
European Journal of Medical Genetics 52 (2009) 337–340
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Short report
Novel BRAF mutation in a patient with LEOPARD syndrome and normal intelligence Monika Koudova a, Eva Seemanova a, Martin Zenker b, * a b
Institute of Biology and Medical Genetics, Charles University, University Hospital Prague, Prague, Czech Republic Institute of Human Genetics, University Hospital of Erlangen, University of Erlangen-Nuremberg, Schwabachanlage 10, 91054 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2009 Accepted 22 April 2009 Available online 3 May 2009
Noonan syndrome (NS) and related disorders are caused by mutations in various genes encoding molecules involved in the RAS–MAPK signalling cascade. There are strong genotype–phenotype correlations. BRAF is the major gene for cardio-facio-cutaneous syndrome (CFCS), and usually patients with a BRAF mutation have significant cognitive impairment. We report on a patient with LEOPARD syndrome and normal intelligence who was found to carry a novel sequence change in BRAF. The mutation p.L245F was demonstrated to be de novo with no evidence of somatic mosaicism. This observation illustrates that the phenotypic spectrum caused by BRAF mutations is broader than previously assumed and that mental retardation is not necessarily associated. We speculate that the impact of p.L245F on BRAF protein function differs either qualitatively or quantitatively from those mutations associated with CFCS. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Noonan syndrome LEOPARD syndrome Cardio-facio-cutaneous syndrome Mitogen-activated protein kinases BRAF
1. Introduction Noonan syndrome (NS; OMIM 163950) and the related disorders such as cardio-facio-cutaneous syndrome (CFCS; OMIM 115150) and LEOPARD syndrome (LS; OMIM 151100) are known to have a common pathogenetic basis in that they are all caused by mutations in molecules of the RAS–MAPK signalling cascade, resulting in dysregulation of this pathway [1]. These entities share many clinical sings including a characteristic spectrum of congenital heart defects, short stature and a similar pattern of cranio-facial anomalies. Nonetheless, some more distinct features usually allow distinguishing LS and CFCS from the more common NS, such as the typical ectodermal anomalies and mental retardation in CFCS, and multiple lentigines and hearing loss in LS. In general, the different phenotypes are strongly correlated with the particular gene/mutation involved: NS is associated with mutations in PTPN11, SOS1, RAF1, while BRAF, MEK1 and MEK2 mutations cause CFCS. In contrast, KRAS mutations appear to have a broader phenotypic spectrum including both, NS and CFCS. LS is consistently associated with specific PTPN11 mutations, but may also be caused by RAF1 mutations. While this rule applies for the vast majority of patients, there have been some recent reports challenging a too strict concept of genotype–phenotype correlations. NS patients with mutations in MEK1 or BRAF were
* Corresponding author. Tel.: þ49 9131 85 22318; fax: þ49 9131 85 23232. E-mail address: [email protected] (M. Zenker). 1769-7212/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2009.04.006
described [2,3], as well as CFCS due to mutated SOS1 [3,4]. Here we report on a patient with a clinical phenotype of LS and high normal intelligence, who was found to carry a novel BRAF mutation. 2. Materials and methods 2.1. Clinical description The patient is a 17-year-old male. He was born after an uneventful pregnancy to healthy unrelated parents of Czech origin. Birth weight at term was 3150 g and body length 51 cm. Paternal and maternal ages at his birth were 40 and 32 years, respectively. Family history was otherwise unremarkable. Tetralogy of Fallot was diagnosed shortly after birth and successful surgical correction was performed at the age of 2 years. Yearly echocardiography controls have been normal, since then. No myocardial hypertrophy has been found. Electrocardiography (ECG) before and after heart surgery showed total right bundle branch block. The patient developed multiple pigmented skin lesions from the age of 5 years. Histology of one of these lesions identified it as a pigmented nevus with epidermal hyperkeratosis. Growth retardation became evident at age 5 years and was first attributed to the cardiac defect. Pubertal development was also delayed, resulting in a maximum deviation of body height from the normal age-related average in the order of magnitude of 2.7 SDS at age 15 years. Bone age at that age was retarded by about 3 years. STH, TSH, LH, FSH, testosterone, and prolactin
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M. Koudova et al. / European Journal of Medical Genetics 52 (2009) 337–340
serum level were found to be normal except for hyperprolactinemia. Treatment with cabergolin was instituted and was associated with some catch-up growth (height SDS at age 17: 2.4). Brain MRI and ophthalmology examination provided no evidence of a pituitary tumor. Only mild myopia (1 diopters) was found. Motor, speech and mental development have been completely normal. Psychological examination using the Wechsler intelligence Scale for Children-Revised (WISC-R) at the age of 12 years revealed a verbal IQ of 121. Total IQ was somewhat lower (110) attributable to mild dyslexia. The boy’s performance is well above average in a grammar school. Unilateral sensorineural highfrequency hearing loss of 30 dB was detected at age 15 years when specifically investigated because of the clinical suspicion of LEOPARD syndrome, but no hearing aids are currently necessary. At clinical examination at the age of 15 years the boy had a body length of 150 cm (2.7 SDS), a weight of 40.7 kg (2.5 SDS) and a head circumference of 58.2 cm (þ1.4 SDS). He displayed facial anomalies typical of the NS/CFCS spectrum. In addition, he had a broad chest with increased intermamillary distance, mild cubitus valgus and curly hair (Fig. 1). A large number of nevi and lentigines were spread over the entire skin. No hyperkeratotic skin changes were present.
2.2. Genetic testing Karyotyping was performed according to standard procedures. DNA was extracted from peripheral blood according to standard procedures and from saliva using the OrageneÒ DNA self collection kit (DNA Genotek, Ottawa, Canada). Molecular testing of PTPN11, SOS1, RAF1, KRAS, BRAF, MEK1, and MEK2 was carried out as described previously [5–8] by direct sequencing using the BigDye terminator method and an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). 3. Results A normal male karyotype was recorded. No mutations were detected in the genes known to cause LS and NS. After expanding our investigations to the genes for CFCS, we identified a sequence change in exon 6 of BRAF (c.735A>G) predicting a novel amino acid substitution p.L245F (Fig. 2). The sequence change was also demonstrated in DNA from buccal cells, thus excluding mosaicism confined to the hematopoetic lineage. De novo occurrence was proven by exclusion of this variation in both parents. Protein sequence alignment indicated that the leucine residue at position
Fig. 1. Clinical photographs of the patient documenting the whole body aspect at age 14 years (a, b) and facial features at age 17 years (c, d). Note hypertelorism, depressed nasal bridge, low-set and posteriorly rotated ears, curly hair, and multiple pigmented skin lesions.
M. Koudova et al. / European Journal of Medical Genetics 52 (2009) 337–340
a Patient (blood) Patient (saliva)
Mother
Father
b BRAF Homo sapiens BRAF Mus musculus BRAF Gallus gallus BRAF Danio rerio BRAF Xenopus laevis Pole hole Apis mellifera Pole hole Drosophila RAF1 Homo sapiens ARAF Homo sapiens
Fig. 2. a, Chromatograms from the patient’s blood and saliva DNA as well as from his parents showing the heterozygous de novo nucleotide exchange. b, Protein sequence alignment of human BRAF and homologs from other species as well as the human paralogs ARAF and CRAF (RAF1). Identical amino acids are shown on black background and similar residues on grey background. Note the complete conservation of the leucine residue at the position corresponding to human Leu-245 (red letters, red arrow). Green arrows indicate amino acid positions known to be mutated in CFCS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
245 is highly conserved through evolution (Fig. 2). The sequence change was not found in more than 300 controls. 4. Discussion The phenotype of this patient appeared to fit best in the category of LS, taking into account the presence of multiple pigmented nevi and lentigines, the patient’s sensorineural hearing impairment, the cardiac conduction defect, and his normal mental development. However, some of the facial features and the patient’s hair texture may be considered rather suggestive of CFCS. Multiple pigmented skin lesions and deafness, though more typical of LEOPARD syndrome, may also occur in CFCS. On the other hand, aside from his curly hair our patient was lacking further ectodermal abnormalities typical of CFCS. These nosologic considerations illustrate the difficulties to reach a definite classification in somewhat atypical cases, since there is no single pathognomonic sign discriminating LS from CFCS. It is becoming more and more evident that the neuro-cardio-facial-cutaneous syndromes represent a clinical spectrum [9] comprising entities that are distinguishable, in general, but whose borders are overlapping. The patient presented here is particularly remarkable for his absence of any cognitive impairment with an IQ even above the mean value, whereas usually patients with BRAF mutations have significant learning disabilities or mental retardation, mostly in the moderate to severe range [2,6,10,11]. Retarded mental development was also mentioned in a patient classified as NS, who harbored the BRAF
339
mutation p.K499E [3]. However, in a study on the cognitive profile in patients with NS, LS and CFCS, Cesarini et al. recently documented one among 10 BRAF mutation-positive patients with an IQ in the normal range (about 85) [12]. The pathogenetic significance of the detected novel BRAF sequence variant is strongly supported by (i) de novo occurrence, (ii) the absence of this change in healthy controls, (iii) a strong conservation of the affected amino acid residue, and (iv) the existence of known mutations affecting amino acid residues neighbouring leucine-245 (T241P, T244P, A246P) (Fig. 2b). These mutations affect a cysteine-rich domain in a region (CR1) highly conserved in RAF proteins, which is involved in RAS binding. The mutant BRAFA246P was found to have increased kinase activity [10], but this does not necessarily mean that p.L245F results in a similar change of functional properties. Considering lack of cognitive impairment in our patient unlike most cases with CFCS, p.L245F might be hypothesized to represent kind of a hypomorph compared to the mutations known to cause CFCS. On the other hand, this patient does not simply display a mild CFCS phenotype. Anyway, it is remarkable that the mutation p.L245F has not been observed before in CFCS patients. This would be in line with the speculation that BRAFL245F might exert specific functional properties that differ from BRAF mutants associated with CFCS, either qualitatively or quantitatively. PTPN11 mutations associated with LS have been shown to have specific consequences on protein function that are distinct from mutations resulting in NS [13,14], pointing at the possibility that LS basically is a phenotype that results from a very specific kind of dysregulated RAS–MAPK signalling. The limitations of this study are related to the fact that this is a single observation. The proposed genotype–phenotype correlations need to be confirmed by additional similar cases. Moreover, although we did not find any evidence of somatic mosaicism by studying DNA from blood and buccal cells, we cannot completely exclude mosaicism confined to tissues not available for genetic testing. In conclusion, this is the first report on LS due to a BRAF mutation. This observation illustrates that the phenotypic spectrum associated with BRAF mutations is broader than previously assumed and documents that mental impairment is not an obligatory consequence of mutations in this gene. While this manuscript was under revision, another group published on novel BRAF mutations associated with NS, LS or CFCS phenotypes [15]. Their study included one case with the same mutation as reported here. This patient was classified as having CFCS on the basis of physical findings, but no information is available regarding his development due to young age (0.3 years). Acknowledgements The authors wish to thank the patient and his family for their kind cooperation and Benedikt Quinger for skilful technical assistance. This work was supported by a grant from the German Research Foundation (DFG; ZE 524/4-1) to M.Z. and IGA-NR 9457-3 to E.S. References [1] Y. Aoki, T. Niihori, Y. Narumi, S. Kure, Y. Matsubara, The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders, Hum. Mutat. 29 (2008) 992–1006. [2] C. Nava, N. Hanna, C. Michot, S. Pereira, N. Pouvreau, T. Niihori, Y. Aoki, Y. Matsubara, B. Arveiler, D. Lacombe, E. Pasmant, B. Parfait, C. Baumann, D. Heron, S. Sigaudy, A. Toutain, M. Rio, A. Goldenberg, B. Leheup, A. Verloes, H. Cave, Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype–phenotype relationships and overlap with Costello syndrome, J. Med. Genet. 44 (2007) 763–771. [3] A.M. Nystrom, S. Ekvall, E. Berglund, M. Bjorkqvist, G. Braathen, K. Duchen, H. Enell, E. Holmberg, U. Holmlund, M. Olsson-Engman, G. Anneren,
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[4]
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M. Koudova et al. / European Journal of Medical Genetics 52 (2009) 337–340 M.L. Bondeson, Noonan and cardio-facio-cutaneous syndromes: two clinically and genetically overlapping disorders, J. Med. Genet. 45 (2008) 500–506. Y. Narumi, Y. Aoki, T. Niihori, M. Sakurai, H. Cave, A. Verloes, K. Nishio, H. Ohashi, K. Kurosawa, N. Okamoto, H. Kawame, S. Mizuno, T. Kondoh, M.C. Addor, A. Coeslier-Dieux, C. Vincent-Delorme, K. Tabayashi, M. Aoki, T. Kobayashi, A. Guliyeva, S. Kure, Y. Matsubara, Clinical manifestations in patients with SOS1 mutations range from Noonan syndrome to CFC syndrome, J. Hum. Genet. 53 (2008) 834–841. S. Schubbert, M. Zenker, S.L. Rowe, S. Boll, C. Klein, G. Bollag, I. van der Burgt, L. Musante, V. Kalscheuer, L.E. Wehner, H. Nguyen, B. West, K.Y. Zhang, E. Sistermans, A. Rauch, C.M. Niemeyer, K. Shannon, C.P. Kratz, Germline KRAS mutations cause Noonan syndrome, Nat. Genet. 38 (2006) 331–336. A.L. Schulz, B. Albrecht, C. Arici, I. van der Burgt, A. Buske, G. Gillessen-Kaesbach, R. Heller, D. Horn, C.A. Hubner, G.C. Korenke, R. Konig, W. Kress, G. Kruger, P. Meinecke, J. Mucke, B. Plecko, E. Rossier, A. Schinzel, A. Schulze, E. Seemanova, H. Seidel, S. Spranger, B. Tuysuz, S. Uhrig, D. Wieczorek, K. Kutsche, M. Zenker, Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome, Clin. Genet. 73 (2008) 62–70. M. Zenker, G. Buheitel, R. Rauch, R. Koenig, K. Bosse, W. Kress, H.U. Tietze, H.G. Doerr, M. Hofbeck, H. Singer, A. Reis, A. Rauch, Genotype–phenotype correlations in Noonan syndrome, J. Pediatr. 144 (2004) 368–374. M. Zenker, D. Horn, D. Wieczorek, J. Allanson, S. Pauli, I. van der Burgt, H.G. Doerr, H. Gaspar, M. Hofbeck, G. Gillessen-Kaesbach, A. Koch, P. Meinecke, S. Mundlos, A. Nowka, A. Rauch, S. Reif, C. von Schnakenburg, H. Seidel, L.E. Wehner, C. Zweier, S. Bauhuber, V. Matejas, C.P. Kratz, C. Thomas, K. Kutsche, SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome, J. Med. Genet. 44 (2007) 651–656.
[9] M. Bentires-Alj, M.I. Kontaridis, B.G. Neel, Stops along the RAS pathway in human genetic disease, Nat. Med. 12 (2006) 283–285. [10] T. Niihori, Y. Aoki, Y. Narumi, G. Neri, H. Cave, A. Verloes, N. Okamoto, R.C. Hennekam, G. Gillessen-Kaesbach, D. Wieczorek, M.I. Kavamura, K. Kurosawa, H. Ohashi, L. Wilson, D. Heron, D. Bonneau, G. Corona, T. Kaname, K. Naritomi, C. Baumann, N. Matsumoto, K. Kato, S. Kure, Y. Matsubara, Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome, Nat. Genet. 38 (2006) 294–296. [11] P. Rodriguez-Viciana, O. Tetsu, W.E. Tidyman, A.L. Estep, B.A. Conger, M.S. Cruz, F. McCormick, K.A. Rauen, Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome, Science 311 (2006) 1287–1290. [12] L. Cesarini, P. Alfieri, F. Pantaleoni, I. Vasta, M. Cerutti, V. Petrangeli, P. Mariotti, C. Leoni, D. Ricci, S. Vicari, A. Selicorni, M. Tartaglia, E. Mercuri, G. Zampino, Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade, Am. J. Med. Genet. A 149A (2009) 140–146. [13] M.I. Kontaridis, K.D. Swanson, F.S. David, D. Barford, B.G. Neel, PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects, J. Biol. Chem. 281 (2006) 6785–6792. [14] K. Oishi, H. Zhang, W.J. Gault, C.J. Wang, C.C. Tan, I.K. Kim, H. Ying, T. Rahman, N. Pica, M. Tartaglia, M. Mlodzik, B.D. Gelb, Phosphatase-defective LEOPARD syndrome mutations in PTPN11 have gain-of-function effects during Drosophila development, Hum. Mol. Genet. (2008). [15] A. Sarkozy, C. Carta, S. Moretti, G. Zampino, M.C. Digilio, F. Pantaleoni, A.P. Scioletti, G. Esposito, V. Cordeddu, F. Lepri, V. Petrangeli, M.L. Dentici, G.M. Mancini, A. Selicorni, C. Rossi, L. Mazzanti, B. Marino, G.B. Ferrero, M.C. Silengo, L. Memo, F. Stanzial, F. Faravelli, L. Stuppia, E. Puxeddu, B.D. Gelb, B. Dallapiccola, M. Tartaglia, Germline BRAF mutations in Noonan, LEOPARD, and cardiofaciocutaneous syndromes: molecular diversity and associated phenotypic spectrum, Hum. Mutat. 30 (2009) 695–702.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Short report
Novel BRAF mutation in a patient with LEOPARD syndrome and normal intelligence Monika Koudova a, Eva Seemanova a, Martin Zenker b, * a b
Institute of Biology and Medical Genetics, Charles University, University Hospital Prague, Prague, Czech Republic Institute of Human Genetics, University Hospital of Erlangen, University of Erlangen-Nuremberg, Schwabachanlage 10, 91054 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2009 Accepted 22 April 2009 Available online 3 May 2009
Noonan syndrome (NS) and related disorders are caused by mutations in various genes encoding molecules involved in the RAS–MAPK signalling cascade. There are strong genotype–phenotype correlations. BRAF is the major gene for cardio-facio-cutaneous syndrome (CFCS), and usually patients with a BRAF mutation have significant cognitive impairment. We report on a patient with LEOPARD syndrome and normal intelligence who was found to carry a novel sequence change in BRAF. The mutation p.L245F was demonstrated to be de novo with no evidence of somatic mosaicism. This observation illustrates that the phenotypic spectrum caused by BRAF mutations is broader than previously assumed and that mental retardation is not necessarily associated. We speculate that the impact of p.L245F on BRAF protein function differs either qualitatively or quantitatively from those mutations associated with CFCS. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Noonan syndrome LEOPARD syndrome Cardio-facio-cutaneous syndrome Mitogen-activated protein kinases BRAF
1. Introduction Noonan syndrome (NS; OMIM 163950) and the related disorders such as cardio-facio-cutaneous syndrome (CFCS; OMIM 115150) and LEOPARD syndrome (LS; OMIM 151100) are known to have a common pathogenetic basis in that they are all caused by mutations in molecules of the RAS–MAPK signalling cascade, resulting in dysregulation of this pathway [1]. These entities share many clinical sings including a characteristic spectrum of congenital heart defects, short stature and a similar pattern of cranio-facial anomalies. Nonetheless, some more distinct features usually allow distinguishing LS and CFCS from the more common NS, such as the typical ectodermal anomalies and mental retardation in CFCS, and multiple lentigines and hearing loss in LS. In general, the different phenotypes are strongly correlated with the particular gene/mutation involved: NS is associated with mutations in PTPN11, SOS1, RAF1, while BRAF, MEK1 and MEK2 mutations cause CFCS. In contrast, KRAS mutations appear to have a broader phenotypic spectrum including both, NS and CFCS. LS is consistently associated with specific PTPN11 mutations, but may also be caused by RAF1 mutations. While this rule applies for the vast majority of patients, there have been some recent reports challenging a too strict concept of genotype–phenotype correlations. NS patients with mutations in MEK1 or BRAF were
* Corresponding author. Tel.: þ49 9131 85 22318; fax: þ49 9131 85 23232. E-mail address: [email protected] (M. Zenker). 1769-7212/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2009.04.006
described [2,3], as well as CFCS due to mutated SOS1 [3,4]. Here we report on a patient with a clinical phenotype of LS and high normal intelligence, who was found to carry a novel BRAF mutation. 2. Materials and methods 2.1. Clinical description The patient is a 17-year-old male. He was born after an uneventful pregnancy to healthy unrelated parents of Czech origin. Birth weight at term was 3150 g and body length 51 cm. Paternal and maternal ages at his birth were 40 and 32 years, respectively. Family history was otherwise unremarkable. Tetralogy of Fallot was diagnosed shortly after birth and successful surgical correction was performed at the age of 2 years. Yearly echocardiography controls have been normal, since then. No myocardial hypertrophy has been found. Electrocardiography (ECG) before and after heart surgery showed total right bundle branch block. The patient developed multiple pigmented skin lesions from the age of 5 years. Histology of one of these lesions identified it as a pigmented nevus with epidermal hyperkeratosis. Growth retardation became evident at age 5 years and was first attributed to the cardiac defect. Pubertal development was also delayed, resulting in a maximum deviation of body height from the normal age-related average in the order of magnitude of 2.7 SDS at age 15 years. Bone age at that age was retarded by about 3 years. STH, TSH, LH, FSH, testosterone, and prolactin
338
M. Koudova et al. / European Journal of Medical Genetics 52 (2009) 337–340
serum level were found to be normal except for hyperprolactinemia. Treatment with cabergolin was instituted and was associated with some catch-up growth (height SDS at age 17: 2.4). Brain MRI and ophthalmology examination provided no evidence of a pituitary tumor. Only mild myopia (1 diopters) was found. Motor, speech and mental development have been completely normal. Psychological examination using the Wechsler intelligence Scale for Children-Revised (WISC-R) at the age of 12 years revealed a verbal IQ of 121. Total IQ was somewhat lower (110) attributable to mild dyslexia. The boy’s performance is well above average in a grammar school. Unilateral sensorineural highfrequency hearing loss of 30 dB was detected at age 15 years when specifically investigated because of the clinical suspicion of LEOPARD syndrome, but no hearing aids are currently necessary. At clinical examination at the age of 15 years the boy had a body length of 150 cm (2.7 SDS), a weight of 40.7 kg (2.5 SDS) and a head circumference of 58.2 cm (þ1.4 SDS). He displayed facial anomalies typical of the NS/CFCS spectrum. In addition, he had a broad chest with increased intermamillary distance, mild cubitus valgus and curly hair (Fig. 1). A large number of nevi and lentigines were spread over the entire skin. No hyperkeratotic skin changes were present.
2.2. Genetic testing Karyotyping was performed according to standard procedures. DNA was extracted from peripheral blood according to standard procedures and from saliva using the OrageneÒ DNA self collection kit (DNA Genotek, Ottawa, Canada). Molecular testing of PTPN11, SOS1, RAF1, KRAS, BRAF, MEK1, and MEK2 was carried out as described previously [5–8] by direct sequencing using the BigDye terminator method and an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). 3. Results A normal male karyotype was recorded. No mutations were detected in the genes known to cause LS and NS. After expanding our investigations to the genes for CFCS, we identified a sequence change in exon 6 of BRAF (c.735A>G) predicting a novel amino acid substitution p.L245F (Fig. 2). The sequence change was also demonstrated in DNA from buccal cells, thus excluding mosaicism confined to the hematopoetic lineage. De novo occurrence was proven by exclusion of this variation in both parents. Protein sequence alignment indicated that the leucine residue at position
Fig. 1. Clinical photographs of the patient documenting the whole body aspect at age 14 years (a, b) and facial features at age 17 years (c, d). Note hypertelorism, depressed nasal bridge, low-set and posteriorly rotated ears, curly hair, and multiple pigmented skin lesions.
M. Koudova et al. / European Journal of Medical Genetics 52 (2009) 337–340
a Patient (blood) Patient (saliva)
Mother
Father
b BRAF Homo sapiens BRAF Mus musculus BRAF Gallus gallus BRAF Danio rerio BRAF Xenopus laevis Pole hole Apis mellifera Pole hole Drosophila RAF1 Homo sapiens ARAF Homo sapiens
Fig. 2. a, Chromatograms from the patient’s blood and saliva DNA as well as from his parents showing the heterozygous de novo nucleotide exchange. b, Protein sequence alignment of human BRAF and homologs from other species as well as the human paralogs ARAF and CRAF (RAF1). Identical amino acids are shown on black background and similar residues on grey background. Note the complete conservation of the leucine residue at the position corresponding to human Leu-245 (red letters, red arrow). Green arrows indicate amino acid positions known to be mutated in CFCS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
245 is highly conserved through evolution (Fig. 2). The sequence change was not found in more than 300 controls. 4. Discussion The phenotype of this patient appeared to fit best in the category of LS, taking into account the presence of multiple pigmented nevi and lentigines, the patient’s sensorineural hearing impairment, the cardiac conduction defect, and his normal mental development. However, some of the facial features and the patient’s hair texture may be considered rather suggestive of CFCS. Multiple pigmented skin lesions and deafness, though more typical of LEOPARD syndrome, may also occur in CFCS. On the other hand, aside from his curly hair our patient was lacking further ectodermal abnormalities typical of CFCS. These nosologic considerations illustrate the difficulties to reach a definite classification in somewhat atypical cases, since there is no single pathognomonic sign discriminating LS from CFCS. It is becoming more and more evident that the neuro-cardio-facial-cutaneous syndromes represent a clinical spectrum [9] comprising entities that are distinguishable, in general, but whose borders are overlapping. The patient presented here is particularly remarkable for his absence of any cognitive impairment with an IQ even above the mean value, whereas usually patients with BRAF mutations have significant learning disabilities or mental retardation, mostly in the moderate to severe range [2,6,10,11]. Retarded mental development was also mentioned in a patient classified as NS, who harbored the BRAF
339
mutation p.K499E [3]. However, in a study on the cognitive profile in patients with NS, LS and CFCS, Cesarini et al. recently documented one among 10 BRAF mutation-positive patients with an IQ in the normal range (about 85) [12]. The pathogenetic significance of the detected novel BRAF sequence variant is strongly supported by (i) de novo occurrence, (ii) the absence of this change in healthy controls, (iii) a strong conservation of the affected amino acid residue, and (iv) the existence of known mutations affecting amino acid residues neighbouring leucine-245 (T241P, T244P, A246P) (Fig. 2b). These mutations affect a cysteine-rich domain in a region (CR1) highly conserved in RAF proteins, which is involved in RAS binding. The mutant BRAFA246P was found to have increased kinase activity [10], but this does not necessarily mean that p.L245F results in a similar change of functional properties. Considering lack of cognitive impairment in our patient unlike most cases with CFCS, p.L245F might be hypothesized to represent kind of a hypomorph compared to the mutations known to cause CFCS. On the other hand, this patient does not simply display a mild CFCS phenotype. Anyway, it is remarkable that the mutation p.L245F has not been observed before in CFCS patients. This would be in line with the speculation that BRAFL245F might exert specific functional properties that differ from BRAF mutants associated with CFCS, either qualitatively or quantitatively. PTPN11 mutations associated with LS have been shown to have specific consequences on protein function that are distinct from mutations resulting in NS [13,14], pointing at the possibility that LS basically is a phenotype that results from a very specific kind of dysregulated RAS–MAPK signalling. The limitations of this study are related to the fact that this is a single observation. The proposed genotype–phenotype correlations need to be confirmed by additional similar cases. Moreover, although we did not find any evidence of somatic mosaicism by studying DNA from blood and buccal cells, we cannot completely exclude mosaicism confined to tissues not available for genetic testing. In conclusion, this is the first report on LS due to a BRAF mutation. This observation illustrates that the phenotypic spectrum associated with BRAF mutations is broader than previously assumed and documents that mental impairment is not an obligatory consequence of mutations in this gene. While this manuscript was under revision, another group published on novel BRAF mutations associated with NS, LS or CFCS phenotypes [15]. Their study included one case with the same mutation as reported here. This patient was classified as having CFCS on the basis of physical findings, but no information is available regarding his development due to young age (0.3 years). Acknowledgements The authors wish to thank the patient and his family for their kind cooperation and Benedikt Quinger for skilful technical assistance. This work was supported by a grant from the German Research Foundation (DFG; ZE 524/4-1) to M.Z. and IGA-NR 9457-3 to E.S. References [1] Y. Aoki, T. Niihori, Y. Narumi, S. Kure, Y. Matsubara, The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders, Hum. Mutat. 29 (2008) 992–1006. [2] C. Nava, N. Hanna, C. Michot, S. Pereira, N. Pouvreau, T. Niihori, Y. Aoki, Y. Matsubara, B. Arveiler, D. Lacombe, E. Pasmant, B. Parfait, C. Baumann, D. Heron, S. Sigaudy, A. Toutain, M. Rio, A. Goldenberg, B. Leheup, A. Verloes, H. Cave, Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype–phenotype relationships and overlap with Costello syndrome, J. Med. Genet. 44 (2007) 763–771. [3] A.M. Nystrom, S. Ekvall, E. Berglund, M. Bjorkqvist, G. Braathen, K. Duchen, H. Enell, E. Holmberg, U. Holmlund, M. Olsson-Engman, G. Anneren,
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