Supporting Online Material for

www.sciencemag.org/cgi/content/full/1124642/dc1 Supporting Online Material for Germline Mutations in Genes within the MAPK Pathway Cause Cardio-facio-cutaneous Syndrome Pablo Rodriguez-Viciana, Osamu Tetsu, William E. Tidyman, Anne L. Estep, Brenda A.Conger, Molly Santa Cruz, Frank McCormick, Katherine A. Rauen* *To whom correspondence should be addressed. E-mail: rauen@cc.ucsf.edu This PDF file includes Materials and Methods Figs. S1 and S2 Tables S1 and S2 References Published 26 January 2006 on Science Express DOI: 10.1126/science.1124642

Supporting Online Material Materials and Methods Patient A cohort of 23 HRAS and PTPN11 mutation-negative individuals with the clinical diagnosis of CFC syndrome participated in this study under an approved institutional review board from the University of California San Francisco. Individuals were either recruited at the 2001 Second International Costello Conference or through the CFC International BioBank (www.cfcsyndrome.org). All patients fulfilled strict diagnostic criteria for CFC including characteristic craniofacial features, cardiac anomalies, characteristic ectodermal and musculoskeletal anomalies, postnatal growth deficiency, hypotonia and developmental delay (table S1). Ages ranged from 1 year 8 months to 23 years. Controls (n=40) were 20 phenotypically unaffected individuals and 20 parents of CFC individuals. Sequence Analysis Genomic DNA was isolated from peripheral blood lymphocytes and buccal cell samples using standard procedures. The entire KRAS, NRAS, MRAS, BRAF, CRAF, MEK1 and MEK2 coding regions were sequenced for mutations using direct, bidirectional sequencing. Exons and intronic flanking regions were amplified by PCR. DNA sequencing was performed using a Big Dye v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer s recommendations and run on an ABI3730xl or ABI3700 capillary sequencing instrument. Data were extracted and analyzed with Sequencer Analysis Software version 3.7 (Applied Biosystems). Buccal cell DNA was used as an alternate tissue source to verify BRAF and MEK1/2 mutations as well as rule out tissue-specific mosaicism (fig. S1). Two individuals in the cohort were found to have no coding mutations in BRAF, MEK1 or MEK2. The promoter, large intronic and 3 untranslated regions were not evaluated for possible molecular alteration which could cause aberrant B-Raf and MEK activity and, therefore, contribute to CFC. In all individuals, no causal mutations were identified in CRAF, KRAS, NRAS and MRAS. GenBank accession numbers are as follows: KRAS (NM 004985), NRAS (NM 002524), MRAS (NM 012219), HRAS (NM 0176795), PTPN11 (NM 002834), BRAF (NM 004333), CRAF (NM 002880), MEK1 (NM 002755), MEK2 (NM 030662). Plasmids and Antibodies Human BRAF cdna with a Myc tag at the 3 end was a kind gift from Martin McMahon. B- Raf was subcloned into pcdna3 with an N-terminal Flag tag. Human MEK2 cdna was purchased from Origene (Rockville, MD) and rat MEK1 cdna (has 99.3% amino acid sequence identity compared to human) was purchased from Upstate (Charlottesville, VA). Each was subcloned into pcdna3 with the human MEK2 insert myc tagged at the 5 end and rat MEK1 insert HA tagged at the 5 end. All mutations were independently introduced using Quick-Change Site-Directed Mutagenesis (Stratagene, La Jolla, CA), and verified by direct sequencing at the UCSF Comprehensive Cancer Center Genome Core Facility. For B-Raf experiments, Myc (9B11),

Phospho-ERK, Total ERK and Phospho-MEK antibodies were purchased from Cell Signaling Technology (Danvers, MA). For the MEK experiments, Myc (A-14) and Phospho-ERK E4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), total ERK V114A was purchased from Promega (Madison, WI) and 12CA5 (HA antibody) was purchased from Roche Diagnostics (Alameda, CA). Transient transfections and Raf assays For B-Raf transfections, human embryonic kidney 293T cells were seeded the day before in six-well dishes and transfected, in duplicate, with 2 µg total plasmid DNA and 5 µl of Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to manufacturer s instructions. Cells were serumstarved (0.5% fetal bovine serum) and 48 hours later lysed in buffer containing Protease and Phosphatase Inhibitor cocktails (Sigma, St. Louis, MO). For MEK1 and MEK2 transient transfections, human embryonic kidney 293 cells were seeded the day before in six-well dishes and transfected, in duplicate, with 2 µg total plasmid DNA and 3 µl of FuGENE 6 (Roche Diagnostics, Alameda, CA). After an overnight incubation, the cells were serum-starved (0.5% fetal bovine serum) and harvested 24 hours later in lysis buffer. MEK and ERK phosphorylation levels were assessed by Western blot. For Raf kinase activity studies, B-Raf was immunoprecipitated with Flag-M2 beads (Sigma, St. Louis, MO). Raf activity was determined in a coupled MEK/ERK2 kinase assay using myelin basic protein (MBP) as the final substrate as previously described (1). Kinase assays were performed in three independent experiments.

Figure S1 Fig. S1. Clinical images and electropherograms from lymphocyte DNA sequencing of MEK1 and MEK2 in probands and their parents. (A) CFC #19 has a F53S MEK1 missense substitution resulting from a nt158t C transition in exon 2. This child has characteristic craniofacial features, ectodermal abnormalities (curly hair, hyperkeratosis, hyperkeratosis pilaris and progressive nevi formation with age), pulmonic stenosis and hypertropic cardiomyopathy, failure to thrive, scoliosis, pectus excavatum, diffuse skeletal demineralization, ocular nystagmus, focal atrophy of the left cerebral hemisphere with prominence of the lateral ventricles, seizures and severe developmental delay. (B) CFC #20 has a Y130C MEK1 substitution in exon 3 resulting from a nt389 A G transition. Although this child has craniofacial dysmorphia consistent with CFC, her features are very mild. Ectodermal anomalies include curly hair, hyperkeratosis, hyperkeratosis pilaris, few nevi and hemangiomas. In addition, she has mild pulmonic stenosis, mild pectus excavatum and mild thinning of the corpus callosum. (C) CFC #21 has a F57C MEK2 missense substitution in exon 2 resulting from a nt170t G transversion. This child has characteristic craniofacial features, ectodermal abnormalities (curly hair, hyperkeratosis, hyperkeratosis pilaris and progressive nevi formation with age), cardiac anomalies (aortic valve defect and nonprogressive ventricular septal hypertrophy), failure to thrive, short stature with growth hormone deficiency, scoliosis and pectus deformity, ocular abnormalities

(nystagmus, strabismus, myopia, bilateral cataracts and optic nerve hypoplasia), cerebellar hypoplasia, prominence of the lateral ventricles, thinning of the corpus callosum and moderate developmental delay. All of these children have hypotonia, heat intolerance and excessive sweating. No mutations were found in MEK1 and MEK2 in parental lymphocyte DNA indicating that the germline mutations in the children had arisen de novo.

Figure S2 Fig. S2. Clinical images and DNA electropherograms of CFC individuals who have BRAF missense mutations that have also been identified in cancer. (A) CFC #16 has an activating F595L B-Raf missense substitution (9). This child has characteristic craniofacial features, ectodermal abnormalities (hyperkeratosis, nevi and curly, sparse hair), a bicuspid aortic valve, failure to thrive, musculoskeletal anomalies (short neck and scoliosis), ocular strabismus, pachygyria, seizures and severe developmental delay. Electropherograms of lymphocyte and buccal DNA demonstrate a nt1785t G transversion. (B) CFC #18 has G469E B-Raf missense mutation within the glycine loop. Although this individual has craniofacial dysmorphia consistent with CFC, unique features include prominence of the supraorbital ridge and cherubism. Cardiac abnormalities include ventricular hypertrophy, atrial septal defect and mitral valve cleft. Ectodermal anomalies are severe and consist of hyperkeratosis, ichthyosis and sparse, curly, friable hair. This individual has ocular and musculoskeletal abnormalities and abnormal brain imaging. Electropherograms of lymphocyte and buccal DNA demonstrate a nt1405g A transition. Fibrous dysplasia of the mandible (cherubism) has been described in patients with NF1 (2) and PTPN11 (3) mutations. One genetic cause of cherubism is heterogeneous missense mutations in exon 9 of SH3BP2, a c-abl binding protein (4). The pathogenesis of cherubism is unknown.

Table S1. Clinical presentation of CFC individuals and germ line mutation analysis of BRAF, MEK1 and MEK2. CFC Individual 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Gene BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF MEK1 MEK1 MEK2 Amino Acid Substitution Q257R S467A E501G Q257R K499E Q257R Q257R E501G N581D G596V E501K N581D Q257R L485F G596V F595L F468S G469E F53S Y130C F57C Sex F M M F M F M M F M F M M M F F F F F F M Age 8yr 9m 6yr 1m 9yr 3m 5yr 2m 10y 9y 9m 3y 8m 18y 5y 9y 1y 8m 3y 8y 2y 23y 4y 6m 10y 21y 15y 7y 12y Ethnicity a C C C C C C C C C C H/C C C C C C C C C C C Characteristic Facies b + + + + + + + + + + + + + + + + + + + - + Developmental Delay c + + + + + + + + + + + + + + + + + + + + + Short stature + + + + + + + + - - - + + - - + + + + + + Cardiac Abnormality d P NL Ectodermal Anomalies e ASD ASD VSD ASD NL NL VSD ASD VSD RD NL NL AV Hair + + + + + + + + + + + + + + + + + + + + + Skin - + - + + + + + + + + - - + + + + + + + + Musculoskeletal Abnormality f + + - + + + + + + + - + + + - + + + + + + GI Abnormality g + - + + + + + - - - - + + + - + + - + + + Ocular Abnormality h + + + + + + + + + - + + + + + + + + + - + Abnormal Brain Image i + + + + + + + + - - - - + * - + + + + + + Seizures - + - - + - + - - - - - + - - + + - + - - Hypotonia + + + + + + + + + + + + + + + + + + + + + Lymphedema/Chylothorax - + - + - - - - - - - + - - - - - - - - - MV TV MV AV a Ethnicity: Caucasian (C), Hispanic (H) b Characteristic facies may include relative macrocephaly, prominent forehead, bitemporal narrowing, shallow orbital ridges, down-slanting palpebral fissures, ptosis, epicanthal folds, short nose with a depressed nasal bridge and low-set, posteriorly rotated ears. c Developmental delay ranges from mild to severe. d Cardiac anomalies may include hypertrophic cardiomyopathy (), peripheral pulmonic stenosis (P), pulmonic stenosis (), atrial septal defect (ASD), ventricular septal defect (VSD), mitral valve dysplasia (MV), tricuspid valve dysplasia (TV), bicuspid aortic valve (AV) or rhythm disturbance (RD); normal (NL). e Ectodermal anomalies may include sparse, curly and/or friable hair, scant eyebrows and eyelashes, hyperkeratosis, hyperkeratosis pilaris and/or ulerythema ophryogenes, nevi, hemangiomas and ichthyosis-like lesions. f Musculoskeletal abnormalities may include short neck, pterygiuim colli, pectus deformity, kyphosis and scoliosis. g Gastrointestinal (GI) abnormalities may include reflux, vomiting or malrotation, requiring NG- or G-tube and/or surgical intervention. h Ophthalmologic abnormalities may include strabismus, nystagmus, astigmatism, myopia, hyperopia and optic nerve hypoplasia. i Brain imaging may have been done by MRI or CT. Abnormalities may include hydrocephalus, ventricular enlargement, cerebral atrophy, pachygyria, agenesis of the corpus callosum or other structural features. * Unknown

Table S2. BRAF mutations in 18 unrelated individuals with CFC syndrome. Germline mutations identified in our cohort are compared with missense mutations that have been reported in cancer. Exon Nucleotide substitution Predicted AA substitution Functional Domain Number of Patients Reported somatic mutations a 6 770A G Q257R protein kinase C 5 no conserved 1 11 1399T G S467A protein kinase 1 no glycine loop 11 1403T C F468S protein kinase 1 F468C glycine loop 11 1405G A G469E protein kinase glycine loop 1 G469E b G469A 12 1455G C L485F protein kinase 1 no 12 1495A G K499E protein kinase 1 no 12 1501G A E501K protein kinase 1 no 12 1502A G E501G protein kinase 2 no 14 1741A G N581D protein kinase 2 N581S catalytic site 15 1785T G F595L protein kinase DFG motif 1 F595L F595S 15 1787G T G596V protein kinase DFG motif 2 G596R a The Sanger Institute Catalogue of Somatic Mutations in Cancer b There have been five reported amino acid substitutions at codon 469, with G469A and G469E being the most frequently reported. G469R, G469S and G469V have been identified less frequently (www.sanger.ac.uk/genetics/cgp/cosmic). Supporting References S1. D. R. Alessi et al., Methods Enzymol. 255, 279 (1995). S2. F. J. Martinez-Tello, P. Manjon-Luengo, M. Martin-Perez, S. Montes-Moreno, Skeletal. Radiol. 34, 793 (2005). S3. T. Jafarov, N. Ferimazova, E. Reichenberger, Clin. Genet. 68, 190 (2005). S4. Y. Ueki et al., Nat. Genet. 28, 125 (2001).