- Email: [email protected]
Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes
Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes Beom Hee Lee, MD,* Jae-Min Kim, MSc,* Hye Young Jin, MD, Gu-Hwan Kim, PhD, Jin-Ho Choi, MD, and Han-Wook Yoo, MD Objectives To investigate mutation spectrums and their correlations to phenotypes in Noonan syndrome (NS) and NS-related disorders that share functional alterations of the Ras-mitogen-activated protein kinase pathway. Study design Clinical characteristics and genotypes of 10 previously known and 2 candidate genes, SPRY1-4 and SPRED1, were investigated in 59 patients with NS, 17 with cardiofaciocutaneous syndrome, 5 with Costello syndrome, and 2 with LEOPARD syndrome. Results PTPN11 (39.0%), SOS1 (20.3%), RAF1 (6.8%), KRAS (5.1%), and BRAF (1.7%) mutations were identified in NS; BRAF (41.2%), SHOC2 (23.5%), and MEK1 (5.9%) mutations in cardiofaciocutaneous syndrome; and HRAS and PTPN11 mutations in Costello syndrome and LEOPARD syndrome, respectively. No additional mutations were identified in 28.9% of NS and 35.3% of cardiofaciocutaneous syndrome. Functional characterizations of 2 RAF1 novel variants, p.P261T and p.S259T, and one SOS1 variant, p.K170E, showed enhanced activity of Rasmitogen-activated protein kinase pathway. Normal stature was frequent in SOS1 mutations, hypertrophic cardiomyopathy in RAF1, and developmental delay in RAF1, BRAF, or SHOC2 mutations. Conclusions By identifying genotype-phenotype correlations, our study highlights the role of molecular genetic testing in the process of differential diagnosis of NS and NS-related disorders. Pathophysiologies that underlie these correlations are needed to be investigated in terms of their effects on Ras-mitogen-activated protein kinase pathway. (J Pediatr 2011;159:1029-35).
N
oonan syndrome (NS; OMIM 163950) is among the most common autosomal dominant growth and developmental disorders.1 LEOPARD syndrome (LS; OMIM 151100), cardiofaciocutaneous syndrome (CFC; OMIM 115150), and Costello syndrome (CS; OMIM 218040) exhibit overlapping phenotypes with NS and are categorized as NS-related disorders.2,3 The molecular genetic pathogenesis of NS and NS-related disorders is related to functional alterations of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway, which is implicated in growth factor–mediated cell proliferation, differentiation, and apoptosis. Gain-of-function germline mutations that affect components of the Ras-MAPK pathway are involved in the development of NS and NS-related disorders.4 PTPN11 (40%-50%), SOS1 (10%-20%), and RAF1 (3%-17%) mutations are common in patients with NS.4-7 A small number of patients with NS can also have KRAS,8 BRAF,9 MEK1,3 or NRAS mutations.10 Recently, SHOC2 mutations were identified in patients with NS-like related disorders with loose anagen hair.11 Still, causative mutations are unknown in 30%-40% of patients with NS.4,7 A total of 60%-80% of patients with CFC have BRAF (approximately 50%), MEK1, and MEK2 mutations.3,9,12-15 PTPN11, RAF1, and BRAF mutations have been described in most patients (95%) with LS, and HRAS mutations are present in most cases with CS.2,9,16,17 Because of these overlapping phenotypic and molecular genetic characteristics among NS and NS-related disorders, clinical delineation of these syndromes can be confusing; however, it is important for proper management as well as genetic counseling. In the current study, the full mutation spectrums of 84 unrelated Korean patients with NS and NS-related disorders were described. To improve the mutation-detection rate, both previously known genes and 2 candidate genes were analyzed with the functional characterizations of the novel variants. With the identification of phenotype-genotype correlations, our study highlights the role of molecular genetic testing in the process of differential diagnosis of NS and NS-related disorders, and discusses their underlying pathophysiologies.
CFC CR2 CS EGF HF LS MAPK NS
Cardiofaciocutaneous syndrome Converted region 2 Costello syndrome Epidermal growth factor Histone-like folds LEOPARD syndrome Mitogen-activated protein kinase Noonan syndrome
From the Department of Pediatrics (B.L., H.J., J.-H.C., H.-W.Y.), Genome Research Center for Birth Defects and Genetic Disorders (B.L., J.-M.K., G.-H.K., H.-W.Y.), Medical Genetics Center (B.L., J.-M.K., G.-H.K., H.W.Y.), Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea *Contributed equally. Supported by a grant from the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Grant No. 2011-0019674). The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2011.05.024
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Methods The institutional review board of the Asan Medical Center, Seoul, Korea, approved the study, and written informed consent was obtained from all subjects. A clinical diagnosis of NS was based on van der Burgt criteria.1 Forty-two patients with NS were recruited from the previous report.7 All the patients were Koreans except one Korean-Vietnamese and were examined by a single medical geneticist. The diagnoses of LS, CFC, and CS were based on clinical findings, including characteristic cardiofaciocutaneous findings.2,3,14 Phenotypic features also were evaluated in all the parents of each patient. Routine karyotyping was performed on peripheral blood leukocytes, and the patients with chromosomal aberrations were excluded. Genomic DNA was extracted from peripheral blood leukocytes by using Puregene DNA isolation kits (Gentra, Minneapolis, Minnesota). The coding regions and flanking intergenic sequences of the PTPN11, SOS1, KRAS, HRAS, NRAS, RAF1, BRAF, SHOC2, MEK1, and MEK2 genes were analyzed by using an ABI3130xl Genetic Analyzer (Applied Biosystems, Foster City, California). In patients without a mutation, the coding regions and flanking intergenic sequences of the candidate genes, SPRY1-4 and SPRED1, were analyzed. Genetic testing of either parent was done in 27 families. Wild-type RAF1 and SOS1 complementary DNA were cloned into pcDNA3.1/V5-His and pcDNA3.1/NT-GFP (Invitrogen, Carlsbad, California) vectors, respectively. Mutant constructs were generated by using the polymerase chain reaction–based DpnI-treatment site-directed mutagenesis method and were verified by sequencing. COS-7 cells were cotransfected with expression plasmids for His-tagged wildtype HRAS and GFP-tagged wild-type or mutant SOS1. Serum-starved cells after transfection were stimulated with 10 ng/mL epidermal growth factor (EGF). The assay was performed with an RAS Activation Assay kit (Millipore, Billerica, Massachusetts) according to the manufacturer’s instructions. COS-7 cells were transfected with His-tagged wild-type or mutant RAF1 expression constructs and were stimulated with 10 ng/mL of EGF. RAF1 kinase assays were carried out according to the manufacturer’s instructions (RAF1 Kinase Assay Kit; Millipore). COS-7 cells were transfected with expression vectors encoding His-ERK2 and His-tagged RAF1 or GFP-tagged SOS1 constructs and were stimulated with 10 ng/mL of EGF. Relative Ras, MEK1, ERK phosphorylation ratios were quantified by using LabWorks 4.6 software (UVP Products, Upland, California), and normalized to total Ras, MEK1, ERK expression, respectively. Each analysis was carried out in at least 3 separate transfections. p.K170E, the product of the SOS1 gene mutant, was mapped onto the crystal structure of the ras activator Son of Sevenless (PDB 3KSY) by using PyMol (http://pymol.sourceforge.net). Statistical Analysis Statistical analyses were performed by using SPSS for Windows (version 12.0; SPSS Inc, Chicago, Illinois). Continuous variables were analyzed by using unpaired t tests and Mann1030
Vol. 159, No. 6 Whitney U test, whereas categorical variables were analyzed by using the Fisher exact test. P values less than .05 were considered to be significant.
Results Among the 83 patients, 59 were clinically diagnosed with NS, 17 with CFC, 5 with CS, and 2 with LS. Five patients with NS and one with LS were familial cases. The mean age at diagnosis was 4.6 5.1 years (range, 0.1-26.2 years). Two patients died at age 3 months due to cardiac failure. The remaining 81 patients were followed-up until age 8.7 4.9 years (mean SD) (range, 1.2-27.1 years). Dysmorphic facial features were typical (31/59 [52.5%]) or suggestive (28/59 [47.5%]) in patients with NS.1 Webbed neck was found in 48.3% of patients with NS. Macrocephaly (3/16 [18.8%]); sparse hair (12/17 [70.6%]); sparse eyelashes (11/16 [68.8%]); and cutaneous findings, such as hyperkeratosis (5/16 [31.3%]) and ichthyosis (8/16 [50.0%]), were pronounced in CFC. Curly hair (5/5), coarse face (5/5), and prominent lip (3/5) were seen in CS. Global developmental delay or mental retardation was more common in NSrelated disorders (22.2% vs 100.0%; P < .001). In addition, short stature (less than a third percentile in growth charts, adjusted according to age and sex18) was more frequent in NSrelated disorders (60.7% vs 87.5%; P = .019). Among the 59 patients with NS, 23 (39.0%), 12 (20.3%), 4 (6.8%), 3 (5.1%), and 1(1.7%) had mutations in the PTPN11, SOS1, RAF1, KRAS, and BRAF genes, respectively. Among the 17 patients with CFC, 7 (41.2%), 4 (23.5%), and 1 (5.9%) were found to have BRAF, SHOC2, and MEK1 mutations, respectively. Typical features of CFC were noted in 4 patients with SHOC2 mutations; relative macrocephaly (4 patients), sparse hair (4), loose anagen hair (3), sparse eyebrow (4), hoarse voice (2), deep palmar crease (1), hyperkeratosis or ichthyosis (4), profound short stature with growth hormone deficiency (4), attention-deficient hyperactivity disorder (1), and global developmental delay or mental retardation (4) (Table I). Five patients with CS carried a single mutation (p.G12S) in HRAS, and 2 patients with LS carried a single mutation (p.Y279C) in PTPN11 (Table II). No mutations in NRAS, MEK2, SPRED1, or SPRY1-4 genes were identified in the remaining 16 patients with NS (27.1%) or the 5 patients with CFC (29.4%). One novel mutant of RAF1, p.P261T, as well as another RAF1 mutant (p.S259T) and a SOS1 mutant (p.K170E), which we described in a previous report,7 were functionally characterized. All 3 variants were located at highly conserved residues. The RAF1 mutants were evaluated by assaying the activation status of the downstream effectors, MEK2 and ERK2, in the COS-7 cells expressing these 2 RAF1 mutants. In the presence of an EGF stimulus, the level of phosphorylated MEK1 and ERK2, measured by immunoblotting, was higher in cells expressing either of the 2 RAF1 mutants, p.P261T and p.S259T, than in those expressing wild-type RAF1 (Figure 1). Lee et al
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Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes
Table I. Genotype and phenotype correlations among patients with NS and NS-related disorders PTPN11 (n = 25) Male:Female Cardiac* Pulmonic stenosis* Hypertrophic cardiomyopathy* Ventricular septal defect* Atrial septal defect* Patent ductus arteriosus* Chest wall deformity* Cryptorchidism* Sensorineuronal hearing loss* Juvenile myelomonocytic leukemia* Bleeding tendency* Growth and development Short stature (<3rd)* Height (SDS)z IGF1 (SDS)z
16:9 (64.0%:36.0%) 18/24 (75.0) 11/24 (45.8)† 5/24 (20.8)† 3/24 (12.5) 6/24 (25.0) 7/24 (29.2) 6/24 (25.0) 7/16 (43.8) 5/24 (20.8) 2/24 (8.3) 4/24 (16.7) 17/23 (73.9) 2.6 1.87 (6.40 to 2.85) 1.72 0.88 (3.13 to 0.83)
z
IGF-BP3 (SDS)
Bone age delay (y)z Global developmental delay or mental retardationz
3.38 2.19 (8.92 to 0.34) 2.47 0.70 (1.00 to 3.60) 6/21 (28.6)
†
SOS1 (n = 12) 7:5 (58.3%:41.7%) 10/12 (83.3) 10/12 (83.3)† 2/12 (16.7)† 3/12 (25.0) 4/12 (33.3) 0/12 (0.0) 7/12 (58.3) 3/7 (42.9) 0/11 (0.0) 0/12 (0.0) 0/12 (0.0) 5/12 (41.7)† 1.94 1.37† (5.11 to 0.61) 2.68 0.89 (3.93 to 1.66) 2.83 1.89 (5.41 to 0.89) 0.82 1.77 (1.90 to 3.00) 0/12 (0.0)†
RAF1 (n = 4)
HRAS (n = 5)
BRAF (n = 8)
KRAS (n = 3)
SHOC2 (n = 4)
3:1 4/4 0/4† 4/4† 1/4 1/4 0/4 1/4 1/3 0/4 0/4 0/4
2:3 5/5 1/5† 3/5 0/5 1/5 0/5 1/5 1/2 1/5 0/5 1/5
3:5 7/8 6/8 3/8 1/8 3/8 0/8 3/8 2/3 1/8 0/8 0/7
3:0 3/3 1/3 0/3 0/2 1/3 0/3 2/3 0/3 0/3 0/3 0/3
2:2 3/3 0/3† 1/3 1/3 2/3 0/3 0/4 0/2 0/4 0/4 0/4
4/4 2.31 0.97 (3.11 to 1.23) nd
5/5† 3.87 1.96† (5.97 to 0.85) nd
6/8 1.60 2.48 (4.40 to 2.63) nd
1/2 nd nd
nd
nd
nd
nd
nd
nd
nd
nd
4/4 3.45 0.53 (3.87 to 2.71) 2.26 0.96 (3.05 to 1.08) 1.93 0.68 (2.69 to 1.28) nd
3/3†
4/4†
2/4
†
5/5
†
7/8
ND, not determined. *P < .05. †N/total (%) of subjects. zMean SD.
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Table II. Molecular genetic diagnoses of patients with NS and NS-related disorders Gene
Exon
Nucleotide change
Amino acid change
Domain
Phenotype
No. patients
PTPN11
3 3 3 3 3 4 7 7 8 8 8 12 12 12 12 13 13 4 6 6 6 10 10 10 10 10 10 7 7 7 2 2 2 6 11 11 12 15 2 3 2
c.181G>A c.184T>G c.188A>G c.215C>G c.236A>G c.417G>C c.836A>G c.844A>G c.854T>C c.922A>G c.923A>G c.1381G>A c.1391G>C c.1403C>T c.1471C>T c.1505A>G c.1510A>G c.508A>G* c.797C>A c.806T>G c.806T>C c.1297G>A c.1322G>A c.1642A>C c.1654A>G c.1655G>A c.1656G>C c.770C>T c.775T>A* c.781T>A* c.40G>A c.65A>G c.108A>G c.770A>G 1390G>C c.1406G>A c.1501G>A c.1785T>G c.4A>G c.398A>G c.34G>A
p.D61N p.Y62D p.Y63C p.A72G p.Q79R p.E139D p.Y279C p.I282V p.F285S p.N308S p.N308D p.A461T p.G464A p.T468M p.P491S p.S502L p.M504V p.K170E* p.T266K p.M269R p.M269T p.E433K p.C441Y p.S548R p.R552G p.R552K p.R552S p.S257L p.S259T* p.P261T* p.V14I p.Q22R p.I36M p.Q257R p.G464R p.G469E p.E501K p.F595L p.S2G p.Y130C p.G12S
N-SH2 N-SH2 N-SH2 N-SH2 N-SH2 C-SH2 PTP PTP PTP PTP PTP PTP PTP PTP PTP PTP PTP HF DH DH DH PH PH HL HL HL HL CR2 CR2 CR2 P-loop P-loop P-loop CR1 CR3 CR3 CR3 CR3 Lysine-rich Protein-kinase P-loop
NS NS NS NS NS NS LS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS CFC NS CFC CFC CFC CFC CFC CS
2 2 1 2 2 1 2 1 1 1 3 2 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 1 1 1 4 1 1 1 1 4 1 5
SOS1
RAF1 KRAS BRAF
SHOC2 MEK1 HRAS
C-SH, C-terminal of the src homology domain; CR, conserved region; DH, Dbl homology domain; HL, helical linker; N-SH, N-terminal of the src homology domain; PH, pleckstrin homology domain; PTP, protein tyrosine phosphatase. *Mutations whose functional characteristics were demonstrated in the current study.
The mutated residue in the SOS1 mutant, p.K170E, is located in the histone-like folds (HF) domain, where mutations have rarely been reported in NS (http://www.hgmd.cf.ac.uk; Table II). The functional alteration of p.K170E was demonstrated by investigating EGF-induced RAS and ERK2 activation. The COS-7 cells transiently expressing p.K170E exhibited increased expression of the Ras-GTP complex, the activated form of Ras, than those expressing wild-type SOS1 upon EGF stimulation. The level of phosphorylated ERK2 was also higher in cells expressing p.K170E (Figure 2). Clinical characteristics were compared among patients with PTPN11 (n = 25), SOS1 (n = 12), RAF1 (n = 4), HRAS (n = 5), BRAF (n = 8), KRAS (n = 3), and SHOC2 mutations (n = 4). As summarized in Table I, pulmonary stenosis was more frequently observed in patients with SOS1 mutations (83.3%) than in those with PTPN11 (45.8%; P = .040), RAF1 (0.0%; P = .008), HRAS (20.0%; P = .028), or SHOC2 mutations (0.0%; P =.022), whereas hypertrophic cardiomyopathy was more frequent in 1032
patients with RAF1 (100.0%) than in those with PTPN11 (20.8%; P = .006) or SOS1 mutations (16.7%; P = .009). Juvenile myelomonocytic leukemia and bladder papillary urothelial hyperplasia were noted in 2 patients with PTPN11 mutations and 1 patient with HRAS mutation, respectively. Height SDS was higher in patients with SOS1 mutations than in those with HRAS mutations (1.94 1.37 vs 3.87 1.96; P = .033), and the proportion of patients with short stature (below the third percentile) also was lower in patients with SOS1 mutations (41.7% vs 100%; P = .044). Analyses of growth profiles revealed no significant differences between patients with SOS1 and PTPN11 mutations, and between those with SOS1 mutation and those with RAF1, BRAF, or SHOC2 mutations (Table I). Global developmental delay or mental retardation was significantly less prevalent in patients with PTPN11 (28.6%) or SOS1 mutations (0.0%) than in those with HRAS (100.0%; P = .007 and P < .001 vs PTPN11 and SOS1, respectively), Lee et al
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Figure 1. Functional characterization of RAF1 mutants identified in patients with NS. A, Wild-type RAF1 (WT), RAF1-S257L (positive control of known mutation), and p.S259T and p.P261T were transiently overexpressed in COS-7 cells. Total MEK and phosphorylated MEK (pMEK) after EGF stimulation were detected with anti-MEK (lower row) and anti-pMEK (upper row) antibodies. B, MEK activation was measured as pMEK/MEK ratio. C, Total ERK and phosphorylated ERK (pERK) in COS-7 cells transfected with His-tagged RAF1 and ERK2 and stimulated with EGF were detected with anti-ERK (lower row) and anti-pERK (upper row) antibodies. D, ERK activation was measured as pERK:ERK ratio. +P < .1 and *P < .05 for comparisons between wildtype and mutant protein (2-tailed Student t test).
BRAF (87.5%; P = .010 and P < .001, respectively), KRAS (100.0%; P = .042 and P = .002, respectively), or SHOC2 mutations (100.0%; P = .017 and P = .001, respectively). No correlations with genotype were observed for other clinical findings.
Discussion Differential diagnoses of NS and NS-related disorders are clinically meaningful because their prognosis and management are different; short stature and global developmental delay or mental retardation are more pronounced in patients with NS-related disorders than in patients with NS. Characteristic craniofacial and ectodermal findings, such as sparse hair, sparse eyebrow, ichthyosis, deep palmar crease, and hyperkeratosis, are important clues for the differential diagnosis, but they can be ambiguous as well. Moreover, the differential diagnosis can be difficult in very young patients such as infants and toddlers. Our study, which described the full mutation spectrums of NS and NS-related disorders, indicates that certain mutations are commonly observed in each phenotype, and identification of genotype also can help the process of differential diagnosis. PTPN11 (39.0%) and SOS1 mutations (20.0%) were the common mutations in NS, as previously reported, whereas RAF1, KRAS, and BRAF mutations were less common.5,6,16,19-22 Importantly, the full mutation spectrum of CFC has not been reported.
In our study, BRAF (41.2%) and SHOC2 (23.5%) mutations were the most common mutations in CFC. Additional mutations in MEK1, MEK2, KRAS, and SOS1 also can be identified in a small number of patients with CFC.3,12-15 SHOC2 mutations were previously reported in patients with NS-like features.11 However, in our study, SHOC2 mutations were not identified in patients with NS but in a substantial proportion of patients with CFC, as in a recent report by Komatsuzaki et al,23 which indicated that SHOC2 mutations can be identified in CFC as well as NS, and they might be more common in CFC. CS and LS were characterized by representative mutations in HRAS and PTPN11, respectively. Still, there exists phenotypic heterogeneity even in a small number of patients with a single genotype. As described in a SHOC2 mutation, a BRAF mutation, p.G464R, was previously identified in a patient with CFC3 but in a patient with NS in our report. This phenotypic overlap can be found in other genes, including MEK1, MEK2, and KRAS as well.3 Identification of more specific correlations between genotypes and phenotypes emphasizes the importance of genotype-specific surveillance and management. Pulmonary stenosis and hematologic malignancies are associated with PTPN11 mutations,24,25 whereas hypertrophic cardiomyopathy shows a strong association with RAF1 mutations.7,16,19 In our study, pulmonary stenosis also was associated with SOS1 mutation (83.3%), as recently reported by Kobayashi et al19 (approximately 70%). In addition, solid tumor surveillance is important to patients with HRAS mutations.17 A low
Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes
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Figure 2. Functional characterization of the p.K170E variant identified in NS. A, GFP-tagged wild-type SOS1 (WT), SOS1-R552K (positive control of known mutation), and p.K170E were transiently overexpressed in COS-7 cells with His-tagged HRAS. SOS1 and total RAS in whole-cell lysates (WCL) of EGF-stimulated cells were detected with anti-SOS1 (lower row) and anti-RAS (middle row) antibodies. Ras-GTP was detected with anti-RAS (upper row) antibody by using RAF1-RBD beads. B, Increased expression of Ras-GTP in cells expressing p.K170E (relative activation over vector). C, Total ERK (lower row) and phosphorylated ERK (pERK; upper row) after EGF stimulation were detected in Cos-7 cells transfected with His-tagged SOS1 and ERK2. D, ERK activation was measured as pERK:ERK ratio. +P < .1 and *P < .05 for comparisons between wild-type and mutant protein (2-tailed Student t test).
prevalence (41.7%) of short stature was observed in our patients with SOS1 mutation as in previous reports.21,26 SOS1 may not be directly related to the Janus kinase-signal transducers and activators of transcription pathway, a major signaling pathway induced by growth hormone that is directly linked to PTPN11 and negatively regulated by it.27 When considering the high prevalence of short stature in NSrelated disorders, the relationships between Ras-MAPK proteins besides PTN11 and the signaling pathways induced by growth hormone also should be evaluated. In addition, the high prevalence of global developmental delay and mental retardation in patients with HRAS, BRAF, KRAS, and SHOC2 mutations compared with those with PTPN11 and SOS1 mutations suggest that mental retardation might be influenced by differences in the nature of disease-causing genes in the Ras-MAPK pathway. However, most of our analyses for genotype-phenotype correlations were underpowered due to the small sample size. The functional effects of the 3 variants identified in our cohort, 1 novel and 2 previously described,7 were verified by exploring the downstream effectors in the Ras-MAPK pathway. RAF1 is activated by GTP-bounded RAS and phosphorylates serine residues of MEK. In this process, the conserved region 2 (CR2) domain of RAF1 plays a major role. Kobayashi et al19 demonstrated that the dephosphorylated status of p.S259 in 1034
the CR2 domain is important for the activation of RAF1. Notably, most RAF1 mutations, including all of those in our cohort, are found in the CR2 domain.7,16,19,20 In particular, 2 novel mutations, p.S259T and p.P261T, were located at or near the p.S259 residue. Accordingly, their in vitro activities were higher than those of wild-type RAF1 in the presence of growth factor. However, p.K170E in SOS1 is located in the HF domain, where NS mutations have rarely been reported. The HF domain is predicted to block the allosteric Rasbinding site and prevents Ras activation by SOS1, stabilizing the autoinhibitory conformation of SOS1 in the resting state. In the presence of a growth stimulus, this blocking is unlocked and Ras binds to SOS1 and is activated.28 Gureasko et al28 reported that ionic interactions between p.R552 or p.S548 in the PH-Rem linker and p.D140 and p.D169 in the HF domain are critical for stabilizing this autoinhibitory conformation. p.K170E is located near these ion-pair connections, and substitution of a negatively charged glutamate residue for the positively charged lysine residue is expected to affect this pairing. p.K170E repels the negatively charged p.D169 residue and instead reaches to another positively charged residue p.Q545 in the PH domain. This altered intramolecular interaction is expected to partially disrupt the autoinhibitory conformation, and expose the Ras-binding site, even in the resting state. Consistent with this inference, Lee et al
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December 2011 endogenous expression of the Ras-GTP complex was higher in the COS-7 cells expressing p.K170E than in those expressing wild-type SOS1. Despite extensive efforts to identify disease-causing genes in NS and NS-related disorders, approximately 30% of patients with NS or CFC remain genetically undiagnosed,3,7,12-14 as in our study. We also analyzed the candidate genes, SPRED1 and SPRY1-4, which encode the members of the Sprouty family of proteins that might modulate receptor tyrosine kinase signaling by repressing the Ras-MAPK pathway29 but found no additional mutations. Further study is needed to identify new disease-causing genes in NS with unknown genotypes to fully understand the molecular pathophysiology of the disorder. n We thank the patients and their families for participating in this study. Submitted for publication Dec 31, 2010; last revision received Mar 23, 2011; accepted May 16, 2011. Reprint requests: Han-Wook Yoo, MD, Genome Research Center for Birth Defects and Genetic Diseases, Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, 388-1 PungnapDong, Songpa-Gu, Seoul 138-736, Korea. E-mail: [email protected]
References 1. van der Burgt I, Berends E, Lommen E, van Beersum S, Hamel B, Mariman E. Clinical and molecular studies in a large Dutch family with Noonan syndrome. Am J Med Genet 1994;53:187-91. 2. Digilio MC, Conti E, Sarkozy A, Mingarelli R, Dottorini T, Marino B, et al. Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet 2002;71:389-94. 3. Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, Niihori T, et al. 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 2007;44:763-71. 4. Aoki Y, Niihori T, Narumi Y, Kure S, Matsubara Y. The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat 2008;29:992-1006. 5. Tartaglia M, Gelb BD. Noonan syndrome and related disorders: genetics and pathogenesis. Annu Rev Genomics Hum Genet 2005;6:45-68. 6. Zenker M, Buheitel G, Rauch R, Koenig R, Bosse K, Kress W, et al. Genotype-phenotype correlations in Noonan syndrome. J Pediatr 2004;144:368-74. 7. Ko JM, Kim JM, Kim GH, Yoo HW. PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype-phenotype correlation in Korean patients with Noonan syndrome. J Hum Genet 2008;53:999-1006. 8. Zenker M, Lehmann K, Schulz AL, Barth H, Hansmann D, Koenig R, et al. Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations. J Med Genet 2007;44:131-5. 9. Sarkozy A, Carta C, Moretti S, Zampino G, Digilio MC, Pantaleoni F, et al. Germline BRAF mutations in Noonan, LEOPARD, and cardiofaciocutaneous syndromes: molecular diversity and associated phenotypic spectrum. Hum Mutat 2009;30:695-702. 10. Cirstea IC, Kutsche K, Dvorsky R, Gremer L, Carta C, Horn D, et al. A restricted spectrum of NRAS mutations causes Noonan syndrome. Nat Genet 2010;42:27-9. 11. Cordeddu V, Di Schiavi E, Pennacchio LA, Ma’ayan A, Sarkozy A, Fodale V, et al. Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair. Nat Genet 2009;41:1022-6.
12. Schulz AL, Albrecht B, Arici C, van der Burgt I, Buske A, GillessenKaesbach G, et al. Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome. Clin Genet 2008;73: 62-70. 13. Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, Verloes A, et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet 2006;38:294-6. 14. Narumi Y, Aoki Y, Niihori T, Neri G, Cave H, Verloes A, et al. Molecular and clinical characterization of cardio-facio-cutaneous (CFC) syndrome: overlapping clinical manifestations with Costello syndrome. Am J Med Genet A 2007;143A:799-807. 15. Dentici ML, Sarkozy A, Pantaleoni F, Carta C, Lepri F, Ferese R, et al. Spectrum of MEK1 and MEK2 gene mutations in cardio-faciocutaneous syndrome and genotype-phenotype correlations. Eur J Hum Genet 2009;17:733-40. 16. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, et al. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 2007;39:1007-12. 17. Kerr B, Delrue MA, Sigaudy S, Perveen R, Marche M, Burgelin I, et al. Genotype-phenotype correlation in Costello syndrome: HRAS mutation analysis in 43 cases. J Med Genet 2006;43:401-5. 18. 2007 Korean Children and Adolescents Growth Standard. Korea Center for Disease Control and Prevention, The Korean Pediatric Society, The Committee for the Development of Growth Standard for Korean Children and Adolescents. 19. Kobayashi T, Aoki Y, Niihori T, Cave H, Verloes A, Okamoto N, et al. Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation. Hum Mutat 2010;31:284-94. 20. Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, et al. Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 2007;39:1013-7. 21. Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, Sarkozy A, et al. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 2007;39:75-9. 22. Zenker M, Horn D, Wieczorek D, Allanson J, Pauli S, van der Burgt I, et al. SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome. J Med Genet 2007;44:651-6. 23. Komatsuzaki S, Aoki Y, Niihori T, Okamoto N, Hennekam RC, Hopman S, et al. Mutation analysis of the SHOC2 gene in Noonanlike syndrome and in hematologic malignancies. J Hum Genet 2010; 55:801-9. 24. Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, van der Burgt I, et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002;70:1555-63. 25. Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003;34:148-50. 26. Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, Joshi VA, et al. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet 2007;39:70-4. 27. Stofega MR, Herrington J, Billestrup N, Carter-Su C. Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GHpromoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol Endocrinol 2000;14:1338-50. 28. Gureasko J, Kuchment O, Makino DL, Sondermann H, Bar-Sagi D, Kuriyan J. Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless. Proc Natl Acad Sci U S A 2010; 107:3430-5. 29. Cabrita MA, Christofori G. Sprouty proteins, masterminds of receptor tyrosine kinase signaling. Angiogenesis 2008;11:53-62.
Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes
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N
oonan syndrome (NS; OMIM 163950) is among the most common autosomal dominant growth and developmental disorders.1 LEOPARD syndrome (LS; OMIM 151100), cardiofaciocutaneous syndrome (CFC; OMIM 115150), and Costello syndrome (CS; OMIM 218040) exhibit overlapping phenotypes with NS and are categorized as NS-related disorders.2,3 The molecular genetic pathogenesis of NS and NS-related disorders is related to functional alterations of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway, which is implicated in growth factor–mediated cell proliferation, differentiation, and apoptosis. Gain-of-function germline mutations that affect components of the Ras-MAPK pathway are involved in the development of NS and NS-related disorders.4 PTPN11 (40%-50%), SOS1 (10%-20%), and RAF1 (3%-17%) mutations are common in patients with NS.4-7 A small number of patients with NS can also have KRAS,8 BRAF,9 MEK1,3 or NRAS mutations.10 Recently, SHOC2 mutations were identified in patients with NS-like related disorders with loose anagen hair.11 Still, causative mutations are unknown in 30%-40% of patients with NS.4,7 A total of 60%-80% of patients with CFC have BRAF (approximately 50%), MEK1, and MEK2 mutations.3,9,12-15 PTPN11, RAF1, and BRAF mutations have been described in most patients (95%) with LS, and HRAS mutations are present in most cases with CS.2,9,16,17 Because of these overlapping phenotypic and molecular genetic characteristics among NS and NS-related disorders, clinical delineation of these syndromes can be confusing; however, it is important for proper management as well as genetic counseling. In the current study, the full mutation spectrums of 84 unrelated Korean patients with NS and NS-related disorders were described. To improve the mutation-detection rate, both previously known genes and 2 candidate genes were analyzed with the functional characterizations of the novel variants. With the identification of phenotype-genotype correlations, our study highlights the role of molecular genetic testing in the process of differential diagnosis of NS and NS-related disorders, and discusses their underlying pathophysiologies.
CFC CR2 CS EGF HF LS MAPK NS
Cardiofaciocutaneous syndrome Converted region 2 Costello syndrome Epidermal growth factor Histone-like folds LEOPARD syndrome Mitogen-activated protein kinase Noonan syndrome
From the Department of Pediatrics (B.L., H.J., J.-H.C., H.-W.Y.), Genome Research Center for Birth Defects and Genetic Disorders (B.L., J.-M.K., G.-H.K., H.-W.Y.), Medical Genetics Center (B.L., J.-M.K., G.-H.K., H.W.Y.), Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Korea *Contributed equally. Supported by a grant from the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Grant No. 2011-0019674). The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2011.05.024
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Methods The institutional review board of the Asan Medical Center, Seoul, Korea, approved the study, and written informed consent was obtained from all subjects. A clinical diagnosis of NS was based on van der Burgt criteria.1 Forty-two patients with NS were recruited from the previous report.7 All the patients were Koreans except one Korean-Vietnamese and were examined by a single medical geneticist. The diagnoses of LS, CFC, and CS were based on clinical findings, including characteristic cardiofaciocutaneous findings.2,3,14 Phenotypic features also were evaluated in all the parents of each patient. Routine karyotyping was performed on peripheral blood leukocytes, and the patients with chromosomal aberrations were excluded. Genomic DNA was extracted from peripheral blood leukocytes by using Puregene DNA isolation kits (Gentra, Minneapolis, Minnesota). The coding regions and flanking intergenic sequences of the PTPN11, SOS1, KRAS, HRAS, NRAS, RAF1, BRAF, SHOC2, MEK1, and MEK2 genes were analyzed by using an ABI3130xl Genetic Analyzer (Applied Biosystems, Foster City, California). In patients without a mutation, the coding regions and flanking intergenic sequences of the candidate genes, SPRY1-4 and SPRED1, were analyzed. Genetic testing of either parent was done in 27 families. Wild-type RAF1 and SOS1 complementary DNA were cloned into pcDNA3.1/V5-His and pcDNA3.1/NT-GFP (Invitrogen, Carlsbad, California) vectors, respectively. Mutant constructs were generated by using the polymerase chain reaction–based DpnI-treatment site-directed mutagenesis method and were verified by sequencing. COS-7 cells were cotransfected with expression plasmids for His-tagged wildtype HRAS and GFP-tagged wild-type or mutant SOS1. Serum-starved cells after transfection were stimulated with 10 ng/mL epidermal growth factor (EGF). The assay was performed with an RAS Activation Assay kit (Millipore, Billerica, Massachusetts) according to the manufacturer’s instructions. COS-7 cells were transfected with His-tagged wild-type or mutant RAF1 expression constructs and were stimulated with 10 ng/mL of EGF. RAF1 kinase assays were carried out according to the manufacturer’s instructions (RAF1 Kinase Assay Kit; Millipore). COS-7 cells were transfected with expression vectors encoding His-ERK2 and His-tagged RAF1 or GFP-tagged SOS1 constructs and were stimulated with 10 ng/mL of EGF. Relative Ras, MEK1, ERK phosphorylation ratios were quantified by using LabWorks 4.6 software (UVP Products, Upland, California), and normalized to total Ras, MEK1, ERK expression, respectively. Each analysis was carried out in at least 3 separate transfections. p.K170E, the product of the SOS1 gene mutant, was mapped onto the crystal structure of the ras activator Son of Sevenless (PDB 3KSY) by using PyMol (http://pymol.sourceforge.net). Statistical Analysis Statistical analyses were performed by using SPSS for Windows (version 12.0; SPSS Inc, Chicago, Illinois). Continuous variables were analyzed by using unpaired t tests and Mann1030
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Results Among the 83 patients, 59 were clinically diagnosed with NS, 17 with CFC, 5 with CS, and 2 with LS. Five patients with NS and one with LS were familial cases. The mean age at diagnosis was 4.6 5.1 years (range, 0.1-26.2 years). Two patients died at age 3 months due to cardiac failure. The remaining 81 patients were followed-up until age 8.7 4.9 years (mean SD) (range, 1.2-27.1 years). Dysmorphic facial features were typical (31/59 [52.5%]) or suggestive (28/59 [47.5%]) in patients with NS.1 Webbed neck was found in 48.3% of patients with NS. Macrocephaly (3/16 [18.8%]); sparse hair (12/17 [70.6%]); sparse eyelashes (11/16 [68.8%]); and cutaneous findings, such as hyperkeratosis (5/16 [31.3%]) and ichthyosis (8/16 [50.0%]), were pronounced in CFC. Curly hair (5/5), coarse face (5/5), and prominent lip (3/5) were seen in CS. Global developmental delay or mental retardation was more common in NSrelated disorders (22.2% vs 100.0%; P < .001). In addition, short stature (less than a third percentile in growth charts, adjusted according to age and sex18) was more frequent in NSrelated disorders (60.7% vs 87.5%; P = .019). Among the 59 patients with NS, 23 (39.0%), 12 (20.3%), 4 (6.8%), 3 (5.1%), and 1(1.7%) had mutations in the PTPN11, SOS1, RAF1, KRAS, and BRAF genes, respectively. Among the 17 patients with CFC, 7 (41.2%), 4 (23.5%), and 1 (5.9%) were found to have BRAF, SHOC2, and MEK1 mutations, respectively. Typical features of CFC were noted in 4 patients with SHOC2 mutations; relative macrocephaly (4 patients), sparse hair (4), loose anagen hair (3), sparse eyebrow (4), hoarse voice (2), deep palmar crease (1), hyperkeratosis or ichthyosis (4), profound short stature with growth hormone deficiency (4), attention-deficient hyperactivity disorder (1), and global developmental delay or mental retardation (4) (Table I). Five patients with CS carried a single mutation (p.G12S) in HRAS, and 2 patients with LS carried a single mutation (p.Y279C) in PTPN11 (Table II). No mutations in NRAS, MEK2, SPRED1, or SPRY1-4 genes were identified in the remaining 16 patients with NS (27.1%) or the 5 patients with CFC (29.4%). One novel mutant of RAF1, p.P261T, as well as another RAF1 mutant (p.S259T) and a SOS1 mutant (p.K170E), which we described in a previous report,7 were functionally characterized. All 3 variants were located at highly conserved residues. The RAF1 mutants were evaluated by assaying the activation status of the downstream effectors, MEK2 and ERK2, in the COS-7 cells expressing these 2 RAF1 mutants. In the presence of an EGF stimulus, the level of phosphorylated MEK1 and ERK2, measured by immunoblotting, was higher in cells expressing either of the 2 RAF1 mutants, p.P261T and p.S259T, than in those expressing wild-type RAF1 (Figure 1). Lee et al
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Spectrum of Mutations in Noonan Syndrome and Their Correlation with Phenotypes
Table I. Genotype and phenotype correlations among patients with NS and NS-related disorders PTPN11 (n = 25) Male:Female Cardiac* Pulmonic stenosis* Hypertrophic cardiomyopathy* Ventricular septal defect* Atrial septal defect* Patent ductus arteriosus* Chest wall deformity* Cryptorchidism* Sensorineuronal hearing loss* Juvenile myelomonocytic leukemia* Bleeding tendency* Growth and development Short stature (<3rd)* Height (SDS)z IGF1 (SDS)z
16:9 (64.0%:36.0%) 18/24 (75.0) 11/24 (45.8)† 5/24 (20.8)† 3/24 (12.5) 6/24 (25.0) 7/24 (29.2) 6/24 (25.0) 7/16 (43.8) 5/24 (20.8) 2/24 (8.3) 4/24 (16.7) 17/23 (73.9) 2.6 1.87 (6.40 to 2.85) 1.72 0.88 (3.13 to 0.83)
z
IGF-BP3 (SDS)
Bone age delay (y)z Global developmental delay or mental retardationz
3.38 2.19 (8.92 to 0.34) 2.47 0.70 (1.00 to 3.60) 6/21 (28.6)
†
SOS1 (n = 12) 7:5 (58.3%:41.7%) 10/12 (83.3) 10/12 (83.3)† 2/12 (16.7)† 3/12 (25.0) 4/12 (33.3) 0/12 (0.0) 7/12 (58.3) 3/7 (42.9) 0/11 (0.0) 0/12 (0.0) 0/12 (0.0) 5/12 (41.7)† 1.94 1.37† (5.11 to 0.61) 2.68 0.89 (3.93 to 1.66) 2.83 1.89 (5.41 to 0.89) 0.82 1.77 (1.90 to 3.00) 0/12 (0.0)†
RAF1 (n = 4)
HRAS (n = 5)
BRAF (n = 8)
KRAS (n = 3)
SHOC2 (n = 4)
3:1 4/4 0/4† 4/4† 1/4 1/4 0/4 1/4 1/3 0/4 0/4 0/4
2:3 5/5 1/5† 3/5 0/5 1/5 0/5 1/5 1/2 1/5 0/5 1/5
3:5 7/8 6/8 3/8 1/8 3/8 0/8 3/8 2/3 1/8 0/8 0/7
3:0 3/3 1/3 0/3 0/2 1/3 0/3 2/3 0/3 0/3 0/3 0/3
2:2 3/3 0/3† 1/3 1/3 2/3 0/3 0/4 0/2 0/4 0/4 0/4
4/4 2.31 0.97 (3.11 to 1.23) nd
5/5† 3.87 1.96† (5.97 to 0.85) nd
6/8 1.60 2.48 (4.40 to 2.63) nd
1/2 nd nd
nd
nd
nd
nd
nd
nd
nd
nd
4/4 3.45 0.53 (3.87 to 2.71) 2.26 0.96 (3.05 to 1.08) 1.93 0.68 (2.69 to 1.28) nd
3/3†
4/4†
2/4
†
5/5
†
7/8
ND, not determined. *P < .05. †N/total (%) of subjects. zMean SD.
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Table II. Molecular genetic diagnoses of patients with NS and NS-related disorders Gene
Exon
Nucleotide change
Amino acid change
Domain
Phenotype
No. patients
PTPN11
3 3 3 3 3 4 7 7 8 8 8 12 12 12 12 13 13 4 6 6 6 10 10 10 10 10 10 7 7 7 2 2 2 6 11 11 12 15 2 3 2
c.181G>A c.184T>G c.188A>G c.215C>G c.236A>G c.417G>C c.836A>G c.844A>G c.854T>C c.922A>G c.923A>G c.1381G>A c.1391G>C c.1403C>T c.1471C>T c.1505A>G c.1510A>G c.508A>G* c.797C>A c.806T>G c.806T>C c.1297G>A c.1322G>A c.1642A>C c.1654A>G c.1655G>A c.1656G>C c.770C>T c.775T>A* c.781T>A* c.40G>A c.65A>G c.108A>G c.770A>G 1390G>C c.1406G>A c.1501G>A c.1785T>G c.4A>G c.398A>G c.34G>A
p.D61N p.Y62D p.Y63C p.A72G p.Q79R p.E139D p.Y279C p.I282V p.F285S p.N308S p.N308D p.A461T p.G464A p.T468M p.P491S p.S502L p.M504V p.K170E* p.T266K p.M269R p.M269T p.E433K p.C441Y p.S548R p.R552G p.R552K p.R552S p.S257L p.S259T* p.P261T* p.V14I p.Q22R p.I36M p.Q257R p.G464R p.G469E p.E501K p.F595L p.S2G p.Y130C p.G12S
N-SH2 N-SH2 N-SH2 N-SH2 N-SH2 C-SH2 PTP PTP PTP PTP PTP PTP PTP PTP PTP PTP PTP HF DH DH DH PH PH HL HL HL HL CR2 CR2 CR2 P-loop P-loop P-loop CR1 CR3 CR3 CR3 CR3 Lysine-rich Protein-kinase P-loop
NS NS NS NS NS NS LS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS CFC NS CFC CFC CFC CFC CFC CS
2 2 1 2 2 1 2 1 1 1 3 2 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 1 1 1 4 1 1 1 1 4 1 5
SOS1
RAF1 KRAS BRAF
SHOC2 MEK1 HRAS
C-SH, C-terminal of the src homology domain; CR, conserved region; DH, Dbl homology domain; HL, helical linker; N-SH, N-terminal of the src homology domain; PH, pleckstrin homology domain; PTP, protein tyrosine phosphatase. *Mutations whose functional characteristics were demonstrated in the current study.
The mutated residue in the SOS1 mutant, p.K170E, is located in the histone-like folds (HF) domain, where mutations have rarely been reported in NS (http://www.hgmd.cf.ac.uk; Table II). The functional alteration of p.K170E was demonstrated by investigating EGF-induced RAS and ERK2 activation. The COS-7 cells transiently expressing p.K170E exhibited increased expression of the Ras-GTP complex, the activated form of Ras, than those expressing wild-type SOS1 upon EGF stimulation. The level of phosphorylated ERK2 was also higher in cells expressing p.K170E (Figure 2). Clinical characteristics were compared among patients with PTPN11 (n = 25), SOS1 (n = 12), RAF1 (n = 4), HRAS (n = 5), BRAF (n = 8), KRAS (n = 3), and SHOC2 mutations (n = 4). As summarized in Table I, pulmonary stenosis was more frequently observed in patients with SOS1 mutations (83.3%) than in those with PTPN11 (45.8%; P = .040), RAF1 (0.0%; P = .008), HRAS (20.0%; P = .028), or SHOC2 mutations (0.0%; P =.022), whereas hypertrophic cardiomyopathy was more frequent in 1032
patients with RAF1 (100.0%) than in those with PTPN11 (20.8%; P = .006) or SOS1 mutations (16.7%; P = .009). Juvenile myelomonocytic leukemia and bladder papillary urothelial hyperplasia were noted in 2 patients with PTPN11 mutations and 1 patient with HRAS mutation, respectively. Height SDS was higher in patients with SOS1 mutations than in those with HRAS mutations (1.94 1.37 vs 3.87 1.96; P = .033), and the proportion of patients with short stature (below the third percentile) also was lower in patients with SOS1 mutations (41.7% vs 100%; P = .044). Analyses of growth profiles revealed no significant differences between patients with SOS1 and PTPN11 mutations, and between those with SOS1 mutation and those with RAF1, BRAF, or SHOC2 mutations (Table I). Global developmental delay or mental retardation was significantly less prevalent in patients with PTPN11 (28.6%) or SOS1 mutations (0.0%) than in those with HRAS (100.0%; P = .007 and P < .001 vs PTPN11 and SOS1, respectively), Lee et al
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Figure 1. Functional characterization of RAF1 mutants identified in patients with NS. A, Wild-type RAF1 (WT), RAF1-S257L (positive control of known mutation), and p.S259T and p.P261T were transiently overexpressed in COS-7 cells. Total MEK and phosphorylated MEK (pMEK) after EGF stimulation were detected with anti-MEK (lower row) and anti-pMEK (upper row) antibodies. B, MEK activation was measured as pMEK/MEK ratio. C, Total ERK and phosphorylated ERK (pERK) in COS-7 cells transfected with His-tagged RAF1 and ERK2 and stimulated with EGF were detected with anti-ERK (lower row) and anti-pERK (upper row) antibodies. D, ERK activation was measured as pERK:ERK ratio. +P < .1 and *P < .05 for comparisons between wildtype and mutant protein (2-tailed Student t test).
BRAF (87.5%; P = .010 and P < .001, respectively), KRAS (100.0%; P = .042 and P = .002, respectively), or SHOC2 mutations (100.0%; P = .017 and P = .001, respectively). No correlations with genotype were observed for other clinical findings.
Discussion Differential diagnoses of NS and NS-related disorders are clinically meaningful because their prognosis and management are different; short stature and global developmental delay or mental retardation are more pronounced in patients with NS-related disorders than in patients with NS. Characteristic craniofacial and ectodermal findings, such as sparse hair, sparse eyebrow, ichthyosis, deep palmar crease, and hyperkeratosis, are important clues for the differential diagnosis, but they can be ambiguous as well. Moreover, the differential diagnosis can be difficult in very young patients such as infants and toddlers. Our study, which described the full mutation spectrums of NS and NS-related disorders, indicates that certain mutations are commonly observed in each phenotype, and identification of genotype also can help the process of differential diagnosis. PTPN11 (39.0%) and SOS1 mutations (20.0%) were the common mutations in NS, as previously reported, whereas RAF1, KRAS, and BRAF mutations were less common.5,6,16,19-22 Importantly, the full mutation spectrum of CFC has not been reported.
In our study, BRAF (41.2%) and SHOC2 (23.5%) mutations were the most common mutations in CFC. Additional mutations in MEK1, MEK2, KRAS, and SOS1 also can be identified in a small number of patients with CFC.3,12-15 SHOC2 mutations were previously reported in patients with NS-like features.11 However, in our study, SHOC2 mutations were not identified in patients with NS but in a substantial proportion of patients with CFC, as in a recent report by Komatsuzaki et al,23 which indicated that SHOC2 mutations can be identified in CFC as well as NS, and they might be more common in CFC. CS and LS were characterized by representative mutations in HRAS and PTPN11, respectively. Still, there exists phenotypic heterogeneity even in a small number of patients with a single genotype. As described in a SHOC2 mutation, a BRAF mutation, p.G464R, was previously identified in a patient with CFC3 but in a patient with NS in our report. This phenotypic overlap can be found in other genes, including MEK1, MEK2, and KRAS as well.3 Identification of more specific correlations between genotypes and phenotypes emphasizes the importance of genotype-specific surveillance and management. Pulmonary stenosis and hematologic malignancies are associated with PTPN11 mutations,24,25 whereas hypertrophic cardiomyopathy shows a strong association with RAF1 mutations.7,16,19 In our study, pulmonary stenosis also was associated with SOS1 mutation (83.3%), as recently reported by Kobayashi et al19 (approximately 70%). In addition, solid tumor surveillance is important to patients with HRAS mutations.17 A low
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Figure 2. Functional characterization of the p.K170E variant identified in NS. A, GFP-tagged wild-type SOS1 (WT), SOS1-R552K (positive control of known mutation), and p.K170E were transiently overexpressed in COS-7 cells with His-tagged HRAS. SOS1 and total RAS in whole-cell lysates (WCL) of EGF-stimulated cells were detected with anti-SOS1 (lower row) and anti-RAS (middle row) antibodies. Ras-GTP was detected with anti-RAS (upper row) antibody by using RAF1-RBD beads. B, Increased expression of Ras-GTP in cells expressing p.K170E (relative activation over vector). C, Total ERK (lower row) and phosphorylated ERK (pERK; upper row) after EGF stimulation were detected in Cos-7 cells transfected with His-tagged SOS1 and ERK2. D, ERK activation was measured as pERK:ERK ratio. +P < .1 and *P < .05 for comparisons between wild-type and mutant protein (2-tailed Student t test).
prevalence (41.7%) of short stature was observed in our patients with SOS1 mutation as in previous reports.21,26 SOS1 may not be directly related to the Janus kinase-signal transducers and activators of transcription pathway, a major signaling pathway induced by growth hormone that is directly linked to PTPN11 and negatively regulated by it.27 When considering the high prevalence of short stature in NSrelated disorders, the relationships between Ras-MAPK proteins besides PTN11 and the signaling pathways induced by growth hormone also should be evaluated. In addition, the high prevalence of global developmental delay and mental retardation in patients with HRAS, BRAF, KRAS, and SHOC2 mutations compared with those with PTPN11 and SOS1 mutations suggest that mental retardation might be influenced by differences in the nature of disease-causing genes in the Ras-MAPK pathway. However, most of our analyses for genotype-phenotype correlations were underpowered due to the small sample size. The functional effects of the 3 variants identified in our cohort, 1 novel and 2 previously described,7 were verified by exploring the downstream effectors in the Ras-MAPK pathway. RAF1 is activated by GTP-bounded RAS and phosphorylates serine residues of MEK. In this process, the conserved region 2 (CR2) domain of RAF1 plays a major role. Kobayashi et al19 demonstrated that the dephosphorylated status of p.S259 in 1034
the CR2 domain is important for the activation of RAF1. Notably, most RAF1 mutations, including all of those in our cohort, are found in the CR2 domain.7,16,19,20 In particular, 2 novel mutations, p.S259T and p.P261T, were located at or near the p.S259 residue. Accordingly, their in vitro activities were higher than those of wild-type RAF1 in the presence of growth factor. However, p.K170E in SOS1 is located in the HF domain, where NS mutations have rarely been reported. The HF domain is predicted to block the allosteric Rasbinding site and prevents Ras activation by SOS1, stabilizing the autoinhibitory conformation of SOS1 in the resting state. In the presence of a growth stimulus, this blocking is unlocked and Ras binds to SOS1 and is activated.28 Gureasko et al28 reported that ionic interactions between p.R552 or p.S548 in the PH-Rem linker and p.D140 and p.D169 in the HF domain are critical for stabilizing this autoinhibitory conformation. p.K170E is located near these ion-pair connections, and substitution of a negatively charged glutamate residue for the positively charged lysine residue is expected to affect this pairing. p.K170E repels the negatively charged p.D169 residue and instead reaches to another positively charged residue p.Q545 in the PH domain. This altered intramolecular interaction is expected to partially disrupt the autoinhibitory conformation, and expose the Ras-binding site, even in the resting state. Consistent with this inference, Lee et al
ORIGINAL ARTICLES
December 2011 endogenous expression of the Ras-GTP complex was higher in the COS-7 cells expressing p.K170E than in those expressing wild-type SOS1. Despite extensive efforts to identify disease-causing genes in NS and NS-related disorders, approximately 30% of patients with NS or CFC remain genetically undiagnosed,3,7,12-14 as in our study. We also analyzed the candidate genes, SPRED1 and SPRY1-4, which encode the members of the Sprouty family of proteins that might modulate receptor tyrosine kinase signaling by repressing the Ras-MAPK pathway29 but found no additional mutations. Further study is needed to identify new disease-causing genes in NS with unknown genotypes to fully understand the molecular pathophysiology of the disorder. n We thank the patients and their families for participating in this study. Submitted for publication Dec 31, 2010; last revision received Mar 23, 2011; accepted May 16, 2011. Reprint requests: Han-Wook Yoo, MD, Genome Research Center for Birth Defects and Genetic Diseases, Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, 388-1 PungnapDong, Songpa-Gu, Seoul 138-736, Korea. E-mail: [email protected]
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