Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Am J Med Genet A. 2014 May 28;164(9):2351–2355. doi: 10.1002/ajmg.a.36620

A PTPN11 allele encoding a catalytically impaired SHP2 protein in a patient with a Noonan syndrome phenotype

Jonathan J Edwards 1,2, Simone Martinelli 4, Luca Pannone 4, Ivan Fai-Man Lo 5, Lisong Shi 3, Lisa Edelmann 3, Marco Tartaglia 4, Ho-Ming Luk 5, Bruce D Gelb 1,2
PMCID: PMC4134745  NIHMSID: NIHMS594666  PMID: 24891296

Abstract

The RASopathies are a relatively common group of phenotypically similar and genetically related autosomal dominant genetic syndromes caused by missense mutations affecting genes participating in the RAS/mitogen-activated protein kinase (MAPK) pathway that include Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML, formerly LEOPARD syndrome). NS and NSML can be difficult to differentiate during infancy, but the presence of multiple lentigines, café au lait spots, and specific cardiac defects facilitate the diagnosis. Furthermore, individual PTPN11 missense mutations are highly specific to each syndrome and engender opposite biochemical alterations on the function of SHP-2, the protein product of that gene. Here, we report on a 5-year-old male with two de novo PTPN11 mutations in cis, c.1471C>T (p.Pro491Ser) and c.1492C>T (p.Arg498Trp), which are associated with NS and NSML, respectively. This boy’s phenotype is intermediate between NS and NSML with facial dysmorphism, short stature, mild global developmental delay, pulmonic stenosis and deafness but absence of café au lait spots or lentigines. The double-mutant SHP-2 was found to be catalytically impaired. This raises the question of whether clinical differences between NS and NSML can be ascribed solely to the relative SHP-2 catalytic activity.

Keywords: double mutation, LEOPARD, Noonan, PTPN11

INTRODUCTION

The RASopathies are a group of phenotypically and genetically related autosomal dominant genetic syndromes caused by mutations in genes encoding proteins participating in signaling through the RAS/mitogen-activated protein kinase (MAPK) pathway and include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML, formerly, LEOPARD syndrome), Costello syndrome, and cardiofaciocutaneous syndrome [Rauen, 2013; Tartaglia and Gelb, 2010]. The RASopathies are relatively common, affecting roughly 1:1000 live births, with NS being the most common. Mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP-2, account for 50% of NS and 90% of NSML [Sarkozy et al., 2008; Tartaglia et al., 2011].

NS and NSML share overlapping features including similar facial dysmorphia, hypertrophic cardiomyopathy (HCM) and pulmonic stenosis (PS), and can be challenging to differentiate clinically in infancy [Digilio et al., 2011]. Nonetheless, the two disorders can often be distinguished because patients with NSML, but very few with NS, manifest multiple lentigines, which can be congenital but emerge by age 5 [Digilio et al., 2011; Sarkozy et al., 2008]. In addition, café-au-lait spots are present in ~75% of infants with NSML but are unusual in NS (~10%) [Digilio et al., 2006]. A minority of patients with NSML has sensorineural deafness (10-25%), a finding that is extremely rare in NS. Conversely, 80-90% of individuals with NSML have HCM, which only 20% of those with NS have [Sarkozy et al., 2004; Shaw et al., 2007].

In contrast to the clinical overlap at young age, the individual PTPN11 missense mutations are highly specific to NS and NSML respectively [Tartaglia et al., 2006]. Moreover, the biochemical alterations engendered by the amino acid substitutions associated with NS and NSML differentiate them quite well. PTPN11 mutations causing NS increase SHP-2’s phosphatase activity, either constitutively or in response to phosphotyrosyl activation, and increase RAS/MAPK signaling [Fragale et al., 2004]. In contrast, PTPN11 mutations causing NSML strongly reduce SHP-2’s activity [Hanna et al., 2006; Tartaglia et al., 2006] and have been suggested to result in dominant-negative effects to signaling through the RAS/MAPK pathway [Kontaridis et al., 2006]. What remains less clear is how such dramatically dissimilar biochemical effects produce such remarkably similar phenotypes.

Here, we report on a patient with two de novo PTPN11 mutations in cis affecting exon 13, c.1471C>T and c.1492C>T (p.Pro491Ser and p.Arg498Trp), which are associated with NS and NSML respectively. Biochemically, the double-mutant SHP-2 shows clear loss of phosphatase activity; however, the child’s phenotype is intermediate between NS and NSML.

MATERIALS AND METHODS/RESULTS

Patient Report

This 5-year old Han Chinese male was born to unrelated healthy parents. He was first evaluated by a clinical geneticist at age 2 years 4 months and found to have relative macrocephaly (head circumference 48 cm, 25th centile) with weight and height both below 3rd centile (10.1 kg, 50th centile for age 1 year 2 months and 82 cm, 50th centile for age 1 year 6 months), sensorineural deafness, mild global developmental delay, mild pectus deformity, and bilateral cryptorchidism. This patient also showed mild dysmorphic facial features including hypertelorism (inner canthal, outer canthal, and interpupillary distances all >97th centile), mild ptosis and downslanting palpebral fissures, low-set and posteriorly angulated ears (Fig 1). A thorough dermatologic evaluation confirmed the absence of café au lait spots and lentigines. This patient was re-evaluated at six-month intervals until age 5, but lentigines were never observed. Echocardiography had first been performed at age 2 months demonstrating valvular stenosis with gradient of 40 to 50 mmHg across the lesion. Repeat echocardiography, performed at similar intervals, failed to show progression of the PS or the development of HCM. At the most recent evaluation at age 5, the patient continued to manifest relative macrosomia with persistence of height and weight below 3rd centile. He had developmental delays, achieving 3.5- to 4-year milestones, and was receiving supportive services at school. Overall, this patient had facial dysmorphism and PS, consistent with NSML or NS, and sensorineural deafness, which discriminates towards NSML, but was missing key dermatologic findings that weakened the clinical diagnosis away from NSML.

Figure 1.

Figure 1

Open in a new tab

Mild dysmorphic facial features at 2 years 4 months (A) and 5 years (B and C).

Molecular Genetics

DNA sequence analysis was performed for PTPN11, the most commonly mutated gene for both NSML and NS, and revealed two missense mutations affecting exon 13, c.1471C>T and c.1492C>T, which predicted the p.P491S and p.R498W substitutions, respectively. The p.P491S mutation has previously been associated with NS and the p.R498W allele causes NSML [Lee et al., 2011; Sarkozy et al., 2004]. Analysis of PTPN11 exon 13 in the parents showed normal sequences. Identity testing using single nucleotide polymorphisms (Sequenom iPLEX Pro Sample ID Panel) ruled out non-paternity (probability of paternity > 99.99991%). Thus, we concluded that the proband had two de novo PTPN11 mutations.

To determine if the two PTPN11 mutations had arisen in cis or trans, the proband’s PTPN11 exon 13 was PCR amplified and subcloned (pCR2.1®-TOPO® TA vector, Life Technologies). The inserts of 12 independent clones were sequenced, of which, five showed reference sequence and seven showed both mutations. No clone with only one mutation was observed. Thus, we concluded that the two de novo PTPN11 mutations arose in cis.

To assess the effects of the P491S and R498W substitutions on catalytic function, these mutants, the double mutant and the wild-type enzyme were expressed in bacteria, purified and their phosphatase activities were determined in vitro basally and following stimulation with the protein tyrosine phosphatase non-receptor type substrate 1 (PTPNS1) bisphosphotyrosyl-containing activation motif (BTAM peptide) [Martinelli et al., 2008]. We then compared the phosphatase activities of the double mutant to wild-type SHP-2 as well as both of the single mutants. As shown in Figure 2, the basal phosphatase activity of the P491S/R498W double mutant protein was barely detectable, similar to the NSML-associated R498W single mutant protein and different from the wild-type and NS-associated P491S single mutant SHP-2 proteins. After BTAM stimulation, the activities of all SHP-2 proteins tested increased significantly. As expected, the NS-associated P491S SHP-2 showed substantially greater phosphatase activity compared to wild-type protein, while the NSML-associated R498W mutant was catalytically impaired, being characterized by a weak response to stimulation. The P491S/R498W double mutant SHP-2 also had reduced phosphatase activity, comparable to the NSML-associated R498W protein. Based on this finding, we concluded that the P491S/R498W double mutant SHP-2 behaved biochemically like an NSML mutant.

Figure 2.

Figure 2

Open in a new tab

In vitro phosphatase activity measured by picomoles of p-nitrophenyl phosphate as a substrate for WT and mutant SHP-2 proteins under basal (white bars) and BTAM stimulated (black bars)conditions. All values represent means and standard deviations from triplicate independent experiments normalized to the basal activity of WT SHP-2. The P491S and R498W mutants exhibited phosphatase activity as expected for NS- and NSML-associated mutant proteins respectively; the P491S/R498W double mutant exhibited phosphatase activity similar to NSML-associated mutations with dramatically reduced basal activity and a muted response to phosphotyrosyl stimulation.

DISCUSSION

In summary, we report on a patient who presented with clinical findings consistent with NS but with sensorineural deafness, an unusual finding. Although the diagnosis of NSML was considered, neither lentigines nor café-au-lait spots were observed. The use of DNA testing, which can often definitively differentiate NS from NSML, was not decisive as the child was found to harbor two PTPN11 mutations—one associated with NS and the other with NSML. Biochemical characterization of the doubly mutant SHP-2 protein resulting from this boy’s in cis mutations was most consistent with the diminished phosphatase activity associated with NSML.

Voron et al. [1976] proposed clinical diagnostic criteria for NSML, such that the presence of multiple lentigines plus two of the other cardinal features of NSML were sufficient for the diagnosis. In the absence of lentigines, it was proposed that patients must have three of the other cardinal features of NSML and have an affected first-degree relative. For our proband, the absence of lentigines would prevent a diagnosis of NSML using the first set of criteria and the fact that his PTPN11 mutations were de novo obviates the possibility of applying the second set of criteria. In the current era where DNA testing is available for patients with RASopathy phenotypes, lentigines are still observed in all but the exceptional person harboring NSML-associated PTPN11 mutations (n.b., to our knowledge, there is no published patient of NSML without lentigines for an individual ≥ 5 years, although there is one allusion to a personal observation of such a patient) [Sarkozy et al., 2008]. Consistent with these considerations, the occurrence of HCM with lentigines and/or café-au-lait spots had been documented in all the subjects reported to carry a heterozygous PTPN11 missense change predicting the p.Arg498Trp amino acid substitution (Table I). For our subject, the lack of lentigines by age 5 as well the absence of café-au-lait spots or HCM make the NSML diagnosis difficult to sustain.

Table I.

Clinical features of individuals carrying PTPN11 mutations at codon 498.

Amino acid
substitution		 Patient Age
(years)		 HCM Multiple
lentigines		 Cafè-au-lait
spots		 Deafness Reference
R498W
1 2 + − + − [Sarkozy et al., 2004]
2a 34 + + − − [Sarkozy et al., 2004]
3 <1 + + + − [Digilio et al., 2006]
4b <1 + NA NA NA [Kratz et al., 2006]
R498L
5 13 + + + − [Sarkozy et al., 2004]
6c 39 − + − + [Du-Thanh et al., 2007]
7 NA NA NA NA NA [Hung et al., 2007]
8 NA NA NA NA NA [Lo et al., 2009]
Open in a new tab

Hypertrophic cardiomyopathy (HCM)

a

Mother of patient 1.

b

This newborn died at 2 months. At the age of 11 days, he exhibited a myeloproliferative disorder with excessively proliferating immature erythroid precursors infiltrating nonhematopoietic organs.

c

This patient was reported to have atrioventricular block.

One limitation of the foregoing is that the phenotypic spectrum of many genetic disorders is expanding as molecular testing is applied more broadly. It is possible that we will discover that some patients with NSML never develop features currently viewed as seminal or do so at older ages.

To date, physicians and scientists studying NS and NSML have struggled to understand how PTPN11 mutations that engender opposite effects on SHP-2’s phosphatase activity can result in such highly similar phenotypes. Mouse models with disease-relevant mutations introduced into the endogenous Ptpn11 gene have begun to reveal the pathogeneses. For instance, mice modeling PTPN11-associated NS typically show substantial hyperactivation of the Mapks, Erk1/2 [Araki et al., 2004]. In contrast, mice modeling PTPN11-associated NSML, which exhibit HCM, show normal Erk1/2 activation in their myocardium but hyperactivation of Akt; pharmacologic blockade of Akt signaling through mTor using rapamycin prevents or reverses the HCM in the NSML mouse [Marin et al., 2011]. Thus, it is becoming apparent that rather different perturbations in signal transduction elicited from specific RASopathy mutations are resulting in comparable organ-level abnormalities.

While one must draw conclusions about the phenotypic effects of this subject’s double PTPN11 missense mutations with caution, as it is only a single patient, this very rare “experiment of nature” does provide some opportunity for novel insight. Since this patient’s double mutant SHP-2 has reduced phosphatase activity, one would have predicted that he would have typical NSML features. Since that is not the case, it implies that perturbations in signal transduction cannot be ascribed simply to the level of SHP-2’s phosphatase activity. Rather, mutant SHP-2 proteins must interact with other signaling proteins in a complex fashion that could depend on several factors. Recently published work aimed at addressing this longstanding paradox presented a dual role for the catalytically inhibiting NSML-associated mutations that, similar to NS-associated mutations, also weaken the autoinhibitory conformation via the interaction of the PTP and N-SH2 domains and instead lead the N-SH2 domain to preferentially interact with scaffolding proteins and growth factor receptors [Yu et al., 2013]. Analogously, we speculate that the P491S/R498W protein is more likely to remain activated after stimulation with phosphotyrosyl binding to its N-SH2 and C-SH2 domains, similar to the P491S single mutant SHP-2 (and other NS-associated mutants). Even though the 2nd mutation, R498W, reduces the phosphatase activity, the double mutant protein presumably exhibits its low-level activity in a temporally inappropriate fashion, which may perturb signaling in a manner more typical for NS. Alternatively, the double mutant SHP-2 may be interacting with other proteins because it is in the active conformation for longer in a manner that is independent of its phosphatase activity.

ACKNOWLEDGMENTS

Two of the authors (B. D. G. and M.T.) are co-inventors for a patent concerning PTPN11 testing for Noonan syndrome and receive royalties for PTPN11 genetic testing. The authors are grateful to the patient and his family for their participation. This work was supported in part by awards from the National Institutes of Health (HL071207) to B.D.G. and Telethon-Italy (GGP13107) to M.T.

REFERENCES

  1. Araki T, Mohi MG, Ismat FA, Bronson RT, Williams IR, Kutok JL, Yang W, Pao LI, Gilliland DG, Epstein JA, Neel BG. Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nature Med. 2004;10:849–857. doi: 10.1038/nm1084. [DOI] [PubMed] [Google Scholar]
  2. Digilio MC, Lepri F, Baban A, Dentici ML, Versacci P, Capolino R, Ferese R, De Luca A, Tartaglia M, Marino B, Dallapiccola B. RASopathies: Clinical Diagnosis in the First Year of Life. Mol Syndromol. 2011;1:282–289. doi: 10.1159/000331266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Digilio MC, Sarkozy A, de Zorzi A, Pacileo G, Limongelli G, Mingarelli R, Calabro R, Marino B, Dallapiccola B. LEOPARD syndrome: clinical diagnosis in the first year of life. Am J of Med Genet Part A. 2006;140:740–746. doi: 10.1002/ajmg.a.31156. [DOI] [PubMed] [Google Scholar]
  4. Du-Thanh A, Cave H, Bessis D, Puso C, Guilhou JJ, Dereure O. A novel PTPN11 gene mutation in a patient with LEOPARD Syndrome. Arch Dermatol. 2007;143:1210–1211. doi: 10.1001/archderm.143.9.1210. [DOI] [PubMed] [Google Scholar]
  5. Fragale A, Tartaglia M, Wu J, Gelb BD. Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation. Hum Mutat. 2004;23:267–277. doi: 10.1002/humu.20005. [DOI] [PubMed] [Google Scholar]
  6. Hanna N, Montagner A, Lee WH, Miteva M, Vidal M, Vidaud M, Parfait B, Raynal P. Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: consequences for PI3K binding on Gab1. FEBS Lett. 2006;580:2477–2482. doi: 10.1016/j.febslet.2006.03.088. [DOI] [PubMed] [Google Scholar]
  7. Hung CS, Lin JL, Lee YJ, Lin SP, Chao MC, Lo FS. Mutational analysis of PTPN11 gene in Taiwanese children with Noonan syndrome. J Formos Med Assoc. 2007;106:169–172. doi: 10.1016/S0929-6646(09)60235-7. [DOI] [PubMed] [Google Scholar]
  8. Kontaridis MI, Swanson KD, David FS, Barford D, Neel BG. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J Biol Chem. 2006;28:6785–6792. doi: 10.1074/jbc.M513068200. [DOI] [PubMed] [Google Scholar]
  9. Kratz CP, Nathrath M, Freisinger P, Dressel P, Assmuss HP, Klein C, Yoshimi A, Burdach S, Niemeyer CM. Lethal proliferation of erythroid precursors in a neonate with a germline PTPN11 mutation. Eur J Pediatr. 2006;165:182–185. doi: 10.1007/s00431-005-0031-x. [DOI] [PubMed] [Google Scholar]
  10. Lee BH, Kim JM, Jin HY, Kim GH, Choi JH, Yoo HW. Spectrum of mutations in Noonan syndrome and their correlation with phenotypes. J Pediatr. 2011;159:1029–1035. doi: 10.1016/j.jpeds.2011.05.024. [DOI] [PubMed] [Google Scholar]
  11. Lo FS, Luo JD, Lee YJ, Shu SG, Kuo MT, Chiou CC. High resolution melting analysis for mutation detection for PTPN11 gene: applications of this method for diagnosis of Noonan syndrome. Clin Chim Acta. 2009;409:75–77. doi: 10.1016/j.cca.2009.08.021. [DOI] [PubMed] [Google Scholar]
  12. Marin TM, Keith K, Davies B, Conner DA, Guha P, Kalaitzidis D, Wu X, Lauriol J, Wang B, Bauer M, et al. Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation. J Clin Invest. 2011;12:1026–1043. doi: 10.1172/JCI44972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Martinelli S, Torreri P, Tinti M, Stella L, Bocchinfuso G, Flex E, Grottesi A, Ceccarini M, Palleschi A, Cesareni G, et al. Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes. Hum Mol Genet. 2008;17:2018–2029. doi: 10.1093/hmg/ddn099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rauen KA. The RASopathies. Annu Rev Genom Hum G. 2013;14:355–369. doi: 10.1146/annurev-genom-091212-153523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sarkozy A, Conti E, Digilio MC, Marino B, Morini E, Pacileo G, Wilson M, Calabro R, Pizzuti A, Dallapiccola B. Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syndrome. J Med Genet. 2004;41:e68. doi: 10.1136/jmg.2003.013466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sarkozy A, Digilio MC, Dallapiccola B. Leopard syndrome. Orphanet J Rare Dis. 2008;3:13. doi: 10.1186/1750-1172-3-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shaw AC, Kalidas K, Crosby AH, Jeffery S, Patton MA. The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child. 2007;92:128–132. doi: 10.1136/adc.2006.104547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tartaglia M, Gelb BD. Disorders of dysregulated signal traffic through the RAS MAPK pathway: phenotypic spectrum and molecular mechanisms. Ann NY Acad Sci. 2010;1214:99–121. doi: 10.1111/j.1749-6632.2010.05790.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tartaglia M, Gelb BD, Zenker M. Noonan syndrome and clinically related disorders. Best Pract Res Cl En. 2011;25:161–179. doi: 10.1016/j.beem.2010.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, Cordeddu V, Zampino G, Burgt I, Palleschi A, Petrucci TC, et al. Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet. 2006;78:279–290. doi: 10.1086/499925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Voron DA, Hatfield HH, Kalkhoff RK. Multiple lentigines syndrome. Case report and review of the literature. Am J Med. 1976;60:447–456. doi: 10.1016/0002-9343(76)90764-6. [DOI] [PubMed] [Google Scholar]
  22. Yu ZH, Xu J, Walls CD, Chen L, Zhang S, Zhang R, Wu L, Wang L, Liu S, Zhang ZY. Structural and mechanistic insights into LEOPARD syndrome-associated SHP2 mutations. J Biol Chem. 2013;288:10472–10482. doi: 10.1074/jbc.M113.450023. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES