Germline mutation in BRAF codon 600 is compatible with human development: de novo p.v600g mutation identified in a patient with CFC syndrome

Transcription

1 Clin Genet 2011: 79: Printed in Singapore. All rights reserved Short Report 2010 John Wiley & Sons A/S CLINICAL GENETICS doi: /j x Germline mutation in BRAF codon 600 is compatible with human development: de novo p.v600g mutation identified in a patient with CFC syndrome Champion KJ, Bunag C, Estep AL, Jones JR, Bolt CH, Rogers RC, Rauen KA, Everman DB. Germline mutation in BRAF codon 600 is compatible with human development: de novo p.v600g mutation identified in a patient with CFC syndrome. Clin Genet 2011: 79: John Wiley & Sons A/S, 2010 BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio-facio-cutaneous (CFC) syndrome. One recurrent somatic mutation, p.v600e, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.v600g), leads to increased ERK and ELK phosphorylation compared to wild-type BRAF but is less strongly activating than the cancer-associated p.v600e mutation. Conflict of interest None. KJ Champion a, C Bunag b, AL Estep b, JR Jones a, CH Bolt a, RC Rogers a, KA Rauen b,c and DB Everman a a Greenwood Genetic Center, Greenwood, SC, USA, b Department of Pediatrics, University of California, San Francisco, CA, USA, and c UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA Key words: BRAF cardio-facio-cutaneous syndrome germline Ras/MAPK pathway RASopathy signal transduction p.v600e p.v600g Corresponding authors: Katherine A. Rauen, MD, PhD, UCSF Helen Diller Family Comprehensive Cancer Center, 2340 Sutter Street, Room S429, San Francisco, CA 94115, USA. Tel.: (415) ; fax: (415) ; rauenk@peds.ucsf.edu David B. Everman, MD, Greenwood Genetic Center, 101 Gregor Mendel Circle, Greenwood, SC 29646, USA. Tel.: (864) ; fax: (864) ; deverman@ggc.org Received 9 February 2010, revised and accepted for publication 23 June 2010 Raf is a serine and threonine protein kinase and mediates the mitogen-activated protein kinase (MAPK) pathway, which is one of the many direct downstream cascades of Ras, a small G-protein [for review see (1)]. BRAF is a well studied oncoprotein with somatic mutations in BRAF occurring in approximately 8% of all human cancers (2). Somatic mutations in BRAF have been reported at a high frequency in melanoma and thyroid cancer and at a lower frequency in colorectal and ovarian cancer. The majority of somatic BRAF mutations result in missense substitutions found in, but not limited to, the glycine-rich loop and the activation segment 468

2 p.v600g mutation in CFC syndrome in the kinase domain. One mutation, p.v600e, accounts for over 90% of somatic BRAF mutations identified in human cancer (2). Germline BRAF mutations have been identified as one of the causes of cardio-facio-cutaneous (CFC) syndrome. CFC syndrome is one of the RASopathies and is caused by dysregulation of activation in the MAPK pathway [for review see (3)]. Heterozygous mutations in four of the known genes that encode proteins of the Ras/MAPK pathway have been associated with CFC syndrome: BRAF (4, 5), MEK1 and MEK2 (4) and KRAS (5). The phenotypic features associated with CFC syndrome include characteristic facies, short stature, and cardiac, ectodermal, gastrointestinal, ocular and musculoskeletal abnormalities [for review see (6)]. Variable neurologic and cognitive abnormalities are universally present and include hypotonia, motor delay, speech delay and/or learning disability (7). To date, neither the common cancer-associated BRAF p.v600e mutation nor any other amino acid substitution of the BRAF V600 codon has been reported in an individual with CFC syndrome. Here, we present a young female with CFC syndrome who harbors a de novo germline mutation involving codon 600 of BRAF. Functional analysis of the novel p.v600g missense mutation shows that this mutation is activating in vitro, albeit to a lesser degree than the cancer-associated p.v600e mutation. Materials and methods Clinical report A Caucasian female was seen for clinical genetic evaluation due to a history of developmental delay. She was conceived via in vitro fertilization by a healthy 33-year-old G 2 P 0 mother and a healthy 32-year-old father. Following an uncomplicated pregnancy and delivery at 33 weeks due to preterm labor, she stayed in the neonatal intensive care unit for 3 weeks for feeding difficulties. Her medical history was notable for gastroesophageal reflux, spherocytic anemia that resolved, truncal hypotonia, joint hypermobility, sinus infections, allergies, recurrent otitis media, bilateral exotropia, eczema, and slow-growing hair requiring only occasional haircuts. Her developmental milestones were globally delayed, and she required Early Intervention as well as speech, occupational, and physical therapies. She had a normal EEG and cranial MRI. Psychoeducational evaluation revealed cognitive abilities in the low average range. The family history was non-contributory. Physical examination (Fig. 1) revealed weight and height below the 5th centile, head circumference at the 25th 50th centile, a tall forehead with bitemporal narrowing, sparse and curly scalp hair, very sparse eyebrows, pale blue irides, mild exotropia, slightly overfolded superior ear helices, broad and depressed nasal root and bridge, coarse facies, slight depression of the mid-lower sternum, flat and thin toenails, dry skin with hyperkeratotic (a) (b) (c) (d) (e) Fig. 1. Clinical photographs of the proband at ages 3 years 7 months (a) and 5 years 10 months (b e). Note her tall forehead, mild facial coarsening, slightly overfolded ear helices, sparse and curly scalp hair, and sparse eyebrows. 469

3 Champion et al. papules, several pigmented nevi, and hypotonia with associated joint laxity. The neck was neither broad nor webbed, and no abnormalities of the spine, heart, abdomen, genitalia, or extremities were appreciated. An electrocardiogram was normal, and an echocardiogram revealed a patent foramen ovale with a trivial left to right shunt seen only by color doppler. An abdominal ultrasound showed mild renal asymmetry. A high resolution blood chromosome analysis, subtelomere analysis by multiplex ligation-dependent probe amplification (MLPA), and sequencing of selected exons of PTPN11 (exons 2 4, 7, 8, and 13) were all normal. Sequencing and analysis Sequencing of the genes known to cause CFC syndrome was performed for the proband as part of a research protocol approved by the Institutional Review Board of Self Regional Healthcare (Greenwood, SC). Parental informed consent was obtained for the studies. Subsequent parental testing was performed on a clinical diagnostic basis. Genomic DNA extracted from the proband s peripheral blood leukocytes (FlexiGene DNA kit; Qiagen, Valencia, CA) was used to amplify all coding exons and intronic flanking regions of BRAF (NM_ ), MEK1 (NM_ ), MEK2 (NM_ ) and KRAS (NM_ ; NM_ ) (Supporting Table S1). Genomic DNA extracted from the proband s buccal epithelial cells and the parents peripheral blood leukocytes was used for targeted analysis of BRAF exon 15. Bidirectional sequencing was performed using the Big Dye Terminator Cycle Sequencing Kit v3.1 on a 3730 automated sequencer (Applied Biosystems, Foster City, CA). Data were analyzed using Sequencher R 4.5 DNA sequence assembly software (Gene Codes Corporation, Ann Arbor, MI). Functional analysis of detected sequence alterations was performed using two different programs: Sorting Intolerant From Tolerant (SIFT; and PMUT ( which are web-based tools for the annotation of pathological mutations in proteins (8 12). The identified novel alterations were also screened against mutation and SNP databases NCBI (www. ncbi.nlm.nih.gov/snp), Ensembl ( ensembl.org/index.html), COSMIC ( ac.uk/genetics/cgp/cosmic) and the Human Gene Mutation Database (HGMD; ac/validate.php). Control samples included 50 healthy individuals (100 alleles) and 60 CFC individuals (120 alleles). Plasmids Human BRAF cdna was a kind gift from Dr Martin McMahon and was cloned into a pcdna3 vector with a Flag-tag at the N-terminus. The BRAF c.1799 T G transversion was introduced using Quick-Change Site-Directed Mutagenesis (Stratagene, La Jolla, CA) and verified by direct sequencing. Transient transfections, kinase assay and Western blot analysis Human embryonic kidney (HEK) 293T cells were seeded the day before transfection in six-well dishes. The cells were transfected, in three independent experiments, with 2 μg total plasmid DNA and 5 μl of Lipofectamine 200 (Life Technologies, Carlsbad, CA) according to manufacturer s instructions. In addition to the CFC p.v600g BRAF mutant, HEK 293T cells were transiently transfected with the following control plasmids: wild-type BRAF, pcdna3 empty vector, kinase active BRAF mutant p.s365a (13), the common cancer mutant p.v600e (2), the known kinaseimpaired CFC BRAF mutant p.e501g, and the most common CFC BRAF mutant p.q257r (4). Cells were serum-starved (0.5% fetal bovine serum) and, 24 h later, lysed in buffer containing Protease and Phosphatase Inhibitor cocktails (Sigma, St Louis, MO). Expression levels of BRAF-flag, total ERK and phosphorylated ERK (perk) were analyzed by Western blot. To further explore the downstream kinase activity of the novel BRAF p.v600e mutant, kinase assays were performed in triplicate using the p44/42 MAP Kinase Assay Kit (Cell Signaling Technology, #9800, Danvers, MA) according to the manufacturer s protocol with the following minor substitutions: the immunoprecipitation beads (#3510) and the anti-elk-1 antibody (#9186). Anti-flag antibody (F1804) was purchased from Sigma Aldrich (St Louis, MO), perk (E-4) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and p44/42 MAP Kinase antibody (total ERK, #9102) was purchased from Cell Signaling Technology (Danvers, MA). Quantification of the kinase assay was performed using the freely available NIH ImageJ software according to instructions provided on the software s website ( Three independent kinase assay blots were quantified, and the relative intensity of the phospho-elk-1 level compared to that of wild-type BRAF was calculated and plotted for each mutant (Fig. 3b). The standard error of the mean (SEM) is represented by the error bars in Figure 3b. 470

4 p.v600g mutation in CFC syndrome Results Bidirectional sequencing of all of the coding regions of BRAF, MEK1, MEK2, andkras using the proband s lymphocyte genomic DNA revealed a heterozygous T G transversion (c.1799 T G) in exon 15 of BRAF (Fig. 2). This predicts a valine to glycine substitution at amino acid 600 (p.v600g) and was not found in 50 healthy individuals (100 alleles) or in 60 individuals with CFC syndrome (120 alleles). DNA from buccal epithelium also harbored the mutation, suggesting that mosaicism is unlikely. Targeted analysis of parental DNA was normal indicating that the mutation occurred de novo (Fig. 2). Sequence analysis also identified an intronic MEK2 alteration in the proband (c g A), which was also present in the proband s unaffected father, suggesting that this change is a benign variant (data not shown). Both the SIFT and PMUT programs predicted that BRAF p.v600g would be a deleterious substitution. To corroborate this prediction, HEK 293T cells were transiently transfected with appropriate control plasmids and BRAF mutant plasmid. We found that the BRAF p.v600g mutant had increased ERK phosphorylation compared to the level induced by empty vector, wild-type BRAF and the CFC kinase-impaired mutant control (Fig. 3a). In addition, the level of ERK phosphorylation induced by BRAF p.v600g was less than that of the known cancer-associated BRAF p.v600e mutant and the BRAF p.s365a control. However, the phospho-erk level was similar to, if not slightly less than, the level of ERK phosphorylation induced by the most common CFC mutant, BRAF p.q257r. The kinase assay confirmed these results. Phosphorylated ELK-1 of the cancer-associated p.v600e mutant was much stronger in activity than the CFC p.v600g and p.q257r mutants (Fig. 3a,b), indicating that the CFC mutants are active, but not to the same elevated level of the cancer mutant p.v600e. These results indicate that the p.v600g BRAF CFC mutant protein is a hypermorph, but is less active than the common cancer-causing p.v600e mutant in vitro. Discussion We have identified the first reported germline missense mutation in the well-characterized, cancercausing BRAF codon 600 (p.v600g) in a child with CFC syndrome. The proband has characteristic craniofacial dysmorphia, ectodermal anomalies, exotropia, gastroesophageal reflux, postnatal growth deficiency, and hypotonia resulting in gross motor delay. She does not have a cardiac abnormality, as seen in approximately 80% of individuals with CFC syndrome. While the majority of CFC individuals are reported to have moderate to severe intellectual impairment (7), our proband has only a mild degree of cognitive impairment. Currently at age 7, she is in good general health and making steady developmental progress. She is repeating regular kindergarten with resource help (a) (c) (b) (d) Fig. 2. Electropherograms of BRAF exon 15 sequencing. The proband has a nucleotide 1799 T G transition (c.1799 T G) causing a heterozygous missense substitution p.v600g in exon 15. This mutation was detected in leukocytes (a) aswellasin buccal epithelial cells (b), suggesting that the mutation is not mosaic. The p.v600g mutation was not detected in the proband s mother or father, indicating that the mutation is de novo (c, d). 471

5 Champion et al. (a) (b) Fig. 3. Functional characterization of the novel BRAF p.v600g mutant. Human embryonic kidney 293T cells were transiently transfected in triplicate with the BRAF p.v600g mutant plasmid and control plasmids. ERK phosphorylation was assayed in triplicate by Western blotting using phospho-specific antibodies (a). Downstream ELK-1 phosphorylation was assessed in triplicate by kinase assays (a). Quantification of the kinase assays was performed using NIH Image J software (b). The bar graph in panel (b) shows the relative intensities of the phospho-elk-1 levels for different mutations as compared to that of wild-type BRAF. A total of three independent blots were quantified. The standard error of the mean (SEM) is represented by the error bars. The p.v600g BRAF mutant protein had increased ERK phosphorylation compared to the level induced by empty vector, wild-type BRAF and the known CFC kinase-impaired control. The phospho-erk and phospho-elk-1 levels of the p.v600g mutant appeared comparable to those of the common CFC p.q257r BRAF mutant protein. However, the p.v600g BRAF mutant protein was less active than the common cancer p.v600e mutant as indicated by the levels of phospho-erk and phospho-elk-1. This indicates that p.v600g is hypermorphic, but to a lesser extent than the common cancer-associated mutant p.v600e. Flag-tagged BRAF served as a marker for transfection efficiency and total ERK served as a loading control. and receives occupational and speech therapies. She has been started on medication for symptoms of attention deficit disorder. In vitro functional analysis of this CFC mutant protein by Western blotting shows that p.v600g is a hypermorph, but not to the kinase activity level of the cancer-associated p.v600e mutation. p.v600e is the most commonly reported BRAF mutation in cancer, although other codon 600 substitutions, including p.v600g, have been reported, but at a much reduced frequency [ ac.uk/genetics/cgp/cosmic; (14 16)]. However, mutations involving BRAF codon 600 have never been reported in individuals with CFC syndrome. It is unclear whether this is simply a chance occurrence or whether codon 600 in particular may play a more crucial role in BRAF function than other residues more commonly mutated in patients with CFC syndrome. There is evidence to suggest that alteration of codon 600 may be poorly tolerated in development. Conditional knock-in of the p.v600e mutation in mice using the Cre/Lox system results in embryonic lethality (17). Directed expression of the p.v600e mutation in the somatic tissues of mice (spleen and liver) causes a proliferative disorder and bone marrow failure leading to death by 4 weeks of age. Furthermore, expression of endogenous BRAF p.v600e in primary mouse embryonic fibroblasts is sufficient to induce several 472

6 p.v600g mutation in CFC syndrome hallmarks of transformation, including increased proliferation, loss of contact inhibition, and morphological transformation, suggesting that BRAF mutation may be a critical event in the development of many human tumors. The patho-biochemistry of the BRAF V600 codon alteration is well documented in cancer. Oncogenic p.v600e mutations are presumed to mimic phosphorylation within the activation segment of the BRAF kinase domain, thereby overriding its dependence on Ras-mediated phosphorylation and allowing cells to proliferate autonomously (2). The p.v600e mutation within the BRAF kinase domain disrupts the hydrophobic interaction between the glycine-rich loop and the activation segment causing destabilization of the inactive conformation of BRAF. It has been shown in vitro that the p.v600e mutant has between a twofold and a 700-fold increase in kinase activity when compared to wild-type BRAF (4, 18). Unlike other RASopathies such as Noonan syndrome (NS), Costello syndrome (CS) and neurofibromatosis type 1 (NF1), it is unclear whether individuals with CFC are at an increased risk for malignancies. The benign and malignant neoplasms that are observed in CS, NS and NF1 have not been reported in CFC syndrome. Acute lymphoblastic leukemia has been reported in two CFC individuals with BRAF mutations (19, 20) and one individual with a MEK2 mutation (21). One individual with a MEK1 mutation developed hepatoblastoma while on immunosuppressive therapy (22). Consequently, as the functional differences between cancer-associated BRAF mutations and those that result in CFC syndrome are not well understood, it is difficult to predict whether the presence of the BRAF p.v600g mutation in the germline of the patient described here will lead to an increased risk for malignancies. In summary, we have identified a de novo pathogenic p.v600g BRAF mutation in an individual with CFC syndrome. To our knowledge, this is the first report of a germline mutation involving codon 600 of BRAF. The discovery of this mutation in our patient establishes that disruption of codon 600 is compatible with human development and causes CFC syndrome. However, the rarity of germline mutations affecting codon 600 is not completely clear. As this individual, along with the reported cohort of individuals with CFC syndrome, is still relatively young, it is not apparent whether individuals with CFC syndrome in general or only those with mutations in specific BRAF codons may have an increased risk of cancer. Supporting Information The following Supporting information is available for this article: Table S1. BRAF, MEK1, MEK2, andkras sequencing primers. Additional Supporting information may be found in the online version of this article. Please note: Wiley-Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Acknowledgements We thank the family who participated in this study for their outstanding collaboration. This project was supported in part by the South Carolina Department of Disabilities and Special Needs, the Genetics Endowment of South Carolina, NIH/NICHD (HD to K.A.R) and NIH/NCRR UCSF-CTSI Grant Number UL1 RR (to K.A.R). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. References 1. Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev Mol Cell Biol 2004: 5: Davies H, Bignell GR, Cox C et al. Mutations of the BRAF gene in human cancer. Nature 2002: 417: Tidyman WE, Rauen KA. The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 2009: 19: Rodriguez-Viciana P, Tetsu O, Tidyman WE et al. Germline mutations in genes within the MAPK pathway cause cardiofacio-cutaneous syndrome. Science 2006: 311: Niihori T, Aoki Y, Narumi Y et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006: 38: Roberts A, Allanson J, Jadico SK et al. The cardiofaciocutaneous syndrome. J Med Genet 2006: 43: Yoon G, Rosenberg J, Blaser S et al. Neurological complications of cardio-facio-cutaneous syndrome. Dev Med Child Neurol 2007: 49: Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res 2001: 11: Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res 2002: 12: Ng PC, Henikoff S. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res 2003: 31: Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009: 4: Ferrer-Costa C, Gelpi JL, Zamakola L et al. PMUT: a webbased tool for the annotation of pathological mutations on proteins. Bioinformatics 2005: 21: Hmitou I, Druillennec S, Valluet A et al. Differential regulation of BRAF isoforms by phosphorylation and autoinhibitory mechanisms. Mol Cell Biol 2007: 27: Shinozaki M, Fujimoto A, Morton DL et al. Incidence of BRAF oncogene mutation and clinical relevance for primary cutaneous melanomas. Clin Cancer Res 2004: 10: Feng YZ, Shiozawa T, Miyamoto T et al. BRAF mutation in endometrial carcinoma and hyperplasia: correlation with KRAS and p53 mutations and mismatch repair protein expression. Clin Cancer Res 2005: 11:

7 Champion et al. 16. Lin J, Takata M, Murata H et al. Polyclonality of BRAF mutations in acquired melanocytic nevi. J Natl Cancer Inst 2009: 101: Mercer K, Giblett S, Green S et al. Expression of endogenous oncogenic V600E B-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts. Cancer Res 2005: 65: Wan PT, Garnett MJ, Roe SM et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of BRAF. Cell 2004: 116: van Den Berg H, Hennekam RC. Acute lymphoblastic leukaemia in a patient with cardiofaciocutaneous syndrome. J Med Genet 1999: 36: Makita Y, Narumi Y, Yoshida M et al. Leukemia in cardiofacio-cutaneous (CFC) syndrome: a patient with a germline mutation in BRAF proto-oncogene. J Pediatr Hematol Oncol 2007: 29: Rauen KA, Tidyman WE, Estep AL et al. Molecular and functional analysis of a novel MEK2 mutation in cardio-faciocutaneous syndrome: transmission through four generations. Am J Med Genet A 2010: 152A: Al-Rahawan MM, Chute DJ, Sol-Church K et al. Hepatoblastoma and heart transplantation in a patient with cardiofacio-cutaneous syndrome. Am J Med Genet A 2007: 143A: