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Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clinicas, 05403-900 Sao Paulo, Brazil
Address all correspondence and requests for reprints to: Dr. Alexander A. L. Jorge, Hospital das Clinicas, Labaratorio de Hormonios, Avenue Dr. Eneas de Carvalho Aguiar 155 PAMB, 2 Andar Bloco 6, 05403-900 Sao Paulo, Brazil. E-mail: alexj{at}usp.br.
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Abstract |
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Objective: The objective of this study was to evaluate the influence of PTPN11 status on response to human GH (hGH) treatment in NS children with short stature.
Setting: This study was performed at a university hospital.
Design: The study design was to conduct a retrospective analysis of 3 yr of hGH treatment and genotyping of PTPN11 in patients with NS.
Patients: Fourteen NS patients, half of them with PTPN11 mutations in heterozygous state, were studied. At the beginning of treatment, there were no clinical or laboratory differences between groups with and without mutations in the PTPN11 gene.
Intervention: Patients were treated with hGH (47 µg/kg·d).
Main Outcome Measures: The main outcome measures were PTPN11 genotype, change in IGF-I levels, and change in height SD score.
Results: Patients with mutations in PTPN11 presented a significantly smaller increment in IGF-I levels during the treatment compared with patients without mutations (86 ± 67 and 202 ± 93 µg/liter, respectively; P = 0.03). hGH treatment significantly improved growth velocity in both groups, with slightly better results observed in patients without mutations. This was translated into greater gains in height SD score relation to baseline during the 3 yr of treatment in patients without mutations (+1.7 ± 0.1) compared with those with mutations (+0.8 ± 0.4; P < 0.01).
Conclusions: Our findings suggest that the presence of PTPN11 mutations in patients with NS indicates a reduced growth response to long-term hGH treatment.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Apart from facial features and cardiac disease, one of the cardinal signs of NS is short stature (1, 3). The cause of growth impairment is unclear, and trials with recombinant human GH (hGH) administration have been performed. Several studies indicate that short-term hGH therapy (1–4 yr) is able to increase growth velocity (GV) and improve height SD score (SDS) (3, 4, 5, 6).
Some researchers have suggested that treated children exceeded their pretreatment predicted heights (5, 7); however, there are insufficient data about long-term results and effects on final height of children with NS treated with hGH.
Recently, PTPN11 (protein tyrosine phosphatase, nonreceptor type 11; NM_002834), which encodes for Src homology protein tyrosine phosphatase-2 (SHP-2), was identified as one of the genes involved in NS. Missense mutations in PTPN11 have been demonstrated in 33–60% of patients with NS (8, 9, 10, 11, 12). SHP-2 is a ubiquitously expressed cytoplasmic protein, a member of a subfamily of protein tyrosine phosphatases that contains two Src homology 2 (SH2) domains (13). In addition to tyrosine phosphatase actions, SHP-2 may act as an adapter molecule through phosphorylation of a tyrosine residue at its amino terminal, thus working as a docking site for other SH2-containing molecules (14). Both functions, adapter molecule and tyrosine phosphatase, are obviously relevant to signal transduction of growth factors and cytokines.
The mutations identified in PTPN11 in NS patients are predicted to be gain of function changes, and they cause a conformational shift in the equilibrium of the molecule favoring the active conformation of the protein, thus augmenting the capacity of dephosphorylation (8). The exact mechanism by which PTPN11 mutations could be responsible for compromising linear growth is still unknown. The GH/IGF-I axis is essential for normal postnatal growth (15, 16), and both GH and IGF-I exert their actions after binding to specific receptors that phosphorylate several tyrosine residues located in the intracellular domain. Activation of these tyrosines is critical for downstream signaling of GH- and IGF-I-stimulated biological responses. Tyrosine dephosphorylation leads to the physiological interruption of these pathways. Therefore, an increased action of the protein SHP-2 is expected to cause a decrease in the actions of GH (17) and IGF-I (18, 19). Hence, gain of function mutations of the PTPN11 gene found in NS could have a negative effect on the actions of these two hormones and consequently influence the response to treatment with hGH.
In this study we analyzed the PTPN11 gene in patients with well-characterized NS who were treated with recombinant hGH. The effectiveness of therapy was compared with the presence or absence of PTPN11 mutations.
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Patients and Methods |
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Fourteen children with NS (10 males and four females) followed at the Developmental Endocrinology Unit of the Hospital das Clinicas of the University of Sao Paulo, diagnosed according to the criteria of van der Burgt (20), were treated with hGH. All children were born at term after uneventful pregnancies. Birth weight was 2735 ± 534 g, and birth length was 48 ± 1.7 cm. Three patients were born small for gestational age (patients 3, 7, and 12). The mean age at the start of treatment was 12.3 yr, and bone age was 9.8 ± 2.7 yr. On initial evaluation, all subjects presented short stature (height SDS, –3.5 ± 0.9) and appropriate weight [body mass index (BMI) SDS, –0.9 ± 1.2]. Ten of 14 subjects were prepubertal, and seven of them initiated puberty during the treatment period. Patient 13 had just started puberty at the beginning of hGH treatment; she received concomitant GnRH analog therapy for 3.4 yr, and her secondary sexual characteristics regressed to the prepubertal stage. Seven of the 14 patients (50%) had the following cardiac alterations: pulmonary valve stenosis (patients 3, 7, and 8), mild left ventricular hypertrophic cardiomyopathy (patient 12), mitral valve prolapse (patients 2 and 5), and aortic ectasy with thickened mitral valve (patient 10). None of our patients needed drugs for their cardiac alterations when they started treatment. Trials focused on assessing the adverse cardiac effects of hGH therapy in patients with NS have suggested that hGH is a safe therapy (6, 21, 22) even in patients with mild left ventricular hypertrophy (6). One patient (patient 12), who before hGH treatment had mild left ventricular hypertrophy, presented an increase in ventricular wall thickness measurements in the second year of treatment; for this reason, hGH therapy was interrupted. Despite interruption of hGH therapy, his cardiac function deteriorated, and 1 yr after the treatment had been interrupted, he underwent cardiac surgery.
Informed parental consent, patient assent, and approval by the hospital ethics committee were obtained before initiating the studies.
Hormone assays
GH was determined using an immunofluorometric method with monoclonal antibodies (AutoDELFIA, Wallac, Turku, Finland). The assays were standardized with the international standard 80/505 of the World Health Organization, and levels from 0.1–38 ng/ml presented an interassay coefficient of variation less than 10%. Basal or stimulated GH levels above 3.3 ng/ml ruled out GH deficiency based on study of normal children in our laboratory (23). IGF-I was determined by RIA after ethanol extraction (Diagnostic Systems Laboratories, Webster, TX). The interassay coefficient of variation was less than 10% for IGF-I levels from 18–500 mg/liter.
Molecular studies
Genomic DNA was isolated from peripheral blood leukocytes from all patients. The PTPN11 gene of the subjects was studied by amplifying exons 1–15 using specific intronic primers (primer sequences and amplification protocols will be sent upon request). PCR products were directly sequenced by the dideoxy chain termination method using an ABI PRISM BigDye Terminator kit (PerkinElmer, Foster City, CA) and were analyzed by an ABI PRISM Genetic Analyzer 3100 automatic DNA sequencer (PerkinElmer).
Treatment with hGH
All children were evaluated at baseline and every 3 months during hGH treatment. Evaluations were performed at the same hour of the day and included measurements of standing height (mean of three measurements on a Harpenden stadiometer), weight (measured with a digital scale), and pubertal status assessed according to the method of Marshall and Tanner (24, 25). BMI was calculated (weight/height2) and expressed as the SDS. GV was determined after an observation period of at least 6 months. Target height was calculated, (father’s height + mother’s height and +13 cm for boys or –13 cm for girls)/2, and expressed as the SDS. Bone age radiographs were recorded every year and were assessed by two observers based on the method of Greulich and Pyle (26). Treatment was started after a period of at least 6 months of observation of basal GV (except for patient 14, whose extremely short predicted height warranted an early start of treatment). hGH was administered sc in a dose of 33–50 µg/kg·d (0.1–0.15 U/kg·d), and the dose was adjusted according to changes in weight every visit. Fourteen patients were treated for at least 1 yr, 12 for 2 yr, and eight for 3 yr. There were no significant differences between patients with shorter and longer periods of treatment when they started hGH treatment, so they were analyzed together.
Statistical analysis
All results are expressed as the mean ± SD. Height, BMI, and IGF-I levels were expressed as SDSs for age and sex. Correlations were tested with Pearson’s or Spearman’s rank correlation when appropriate. Differences between groups were tested by t test and Fisher’s exact test.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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PTPN11 gene sequencing identified five different missense mutations (N58D, D61N, A72G, N308D, and M504V) in the heterozygous state in seven of the 14 (50%) patients with NS (Table 1
). All of these mutations were de novo mutations confirmed by direct sequencing of the parents’ PTPN11.
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At the beginning of treatment, there were no significant differences between groups with and without mutations in the PTPN11 gene, concerning gender, age, target height, presence of clinical signs or cardiac malformation, pubertal status, height SDS, BMI SDS, and basal growth rate (Table 2). GH deficiency was excluded in all patients by the presence of a normal GH response (peak GH, 13.1 ± 7.1 ng/ml) to a clonidine stimulation test, and there was no difference between patients with and without the PTPN11 mutation regarding the GH peak. Despite normal GH secretion, mean basal IGF-I levels were low in both groups (–2.0 ± 1.4 SD, according to sex and chronological age).
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The mean hGH dose was 47 µg/kg·d (0.14 U/kg·d) and did not differ significantly between patients with and without PTPN11 mutation. Basal IGF-I levels were similar in both groups at the start of treatment. IGF-I increased after the first year on hGH therapy in patients with mutations in PTPN11 from 130 ± 71 to 216 ± 46 µg/liter (IGF-I SDS, –2.2 ± 1.0 to –1.2 ± 0.8), and this increment was significantly lower than the IGF-I increase observed in patients without the mutation, whose IGF-I rose from 133 ± 75 to 324 ± 136 µg/liter (IGF-I SDS, –1.9 ± 1.8 to +0.1 ± 1.5; P = 0.03; Fig. 1). In the second year of treatment, IGF-I levels remained stable in relation to the first year values (mean of 290 µg/liter in patients with the PTPN11 mutation and 334 µg/liter in patients without the mutation).
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). This translated into greater gains in height SDS compared with baseline in patients without PTPN11 mutations compared with that in those with mutations (Fig. 2), reaching significant levels by the end of the third year of treatment (+1.7 ± 0.1 and +0.8 ± 0.4, respectively; P < 0.01). There was a positive correlation between the increment in GV after the first year of hGH therapy and the increment in IGF-I levels (r = 0.67; P = 0.02).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We found five heterozygous mutations (N58D, D61N, A72G, N308D, and M504V) in the PTPN11 gene in seven cases. They represented 50% of our cohort, a frequency similar to that described in previous studies (8, 9, 10, 11, 12). Three of the mutations (50%) are located on exon 3, which encodes for the amino SH2 domain (N-SH2). The others were on exons 8 (33.3%) and 13 (16.7%), responsible for the catalytic tyrosine phosphatase domain (PTP). They grouped in amino acids directly involved in intramolecular binding (N58D, D61N, and A72G) or were located in the N-SH2 and PTP interaction surface (N308D and M504V). All of them had been previously reported (8, 9, 11, 12). Hence, in agreement with other studies, PTPN11 mutations explain only some of the cases of NS, implying that other genes must also be responsible for this disorder. Additionally, there is great intra- and interfamilial variation in the expressivity of the same mutation (9, 28). PTPN11 mutations are positively correlated with the presence of pulmonary valve stenosis (9, 10, 28) and atrial septal defects (10), but contradictory correlations between the presence of PTPN11 mutation and auxological data were reported (9, 10, 12). In our study we evaluated the influence of PTPN11 mutation status on hGH therapy response.
The groups with and without mutations presented similar clinical and hormonal aspects before initiating treatment. Both groups had low pretreatment IGF-I levels, although they had a normal GH response to stimulation test. GH treatment at supraphysiological dose normalized IGF-I levels; however, patients with the mutation presented, on the average, a smaller increase in IGF-I levels during hGH therapy than patients without mutations despite the fact that hGH therapy was performed using the same dose. The increase in IGF-I positively correlated with the GV increase, in agreement with Noordam et al. (27), who also demonstrated a positive correlation between change in height SDS and the increment in IGF-I over the first year of treatment.
In relation to pretreatment, both groups of patients exhibited an increase in GV during hGH treatment (2.5 cm/yr in patients with mutations on PTPN11 and 3.7 cm/yr in patients without mutations). Studies that did not evaluate PTPN11 status showed similar increases in GV as observed in our patients (2.2–4 cm/yr) in the first year of treatment (5, 29, 30, 31) with an attenuated response thereafter (5). In our study throughout the 3 yr of follow-up, patients without mutations presented better GV, which culminated in a significantly better outcome in height SDS at the end of the third year of hGH treatment. The number of patients who reached 3 yr of treatment is still relatively small and heterogeneous. However, all of these findings, the larger increase in IGF-I levels and the greater long-term height gain in patients without PTPN11 mutation, albeit the fact that they received similar hGH doses as patients with mutations, suggest that PTPN11 mutations can have a negative influence on long-term therapy with GH, possibly by interfering with GH signaling.
The advance in bone age observed in our patients during hGH treatment was noteworthy. Similarly, Noordam et al. (27), evaluating the effect of hGH on linear growth and bone maturation of 37 subjects with NS, noticed that mean bone maturation was significantly faster in the GH treatment group (1.2 vs. 0.5 yr/yr) and seemed to compromise the final height prognosis. Bone age advance was similar in patients with and without PTPN11 mutations in our study.
This is the first time that a correlation between the presence of mutations in the PTPN11 gene and the response to hGH was attempted. Patients with mutations presented a lower increase in IGF-I levels during hGH treatment and presented a significantly lower gain in height SDS after 3 yr of GH treatment compared with patients without mutations. These findings need to be confirmed in additional studies involving a larger number of patients treated for a prolonged period until final height is achieved.
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Footnotes |
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First Published Online June 14, 2005
Abbreviations: BMI, Body mass index; GV, growth velocity; hGH, human GH; NS, Noonan syndrome; PTPN11, protein tyrosine phosphatase, nonreceptor type 11; SDS, SD score; SH2, Src homology 2; SHP-2, Src homology protein tyrosine phosphatase-2.
Received December 30, 2004.
Accepted June 7, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Vß3 integrin regulates insulin-like growth factor I (IGF-I) receptor phosphorylation by altering the rate of recruitment of the Src-homology 2-containing phosphotyrosine phosphatase-2 to the activated IGF-I receptor. Endocrinology 143:4259–4264This article has been cited by other articles:
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  C Noordam, P G M Peer, I Francois, J De Schepper, I van den Burgt, and B J Otten Long-term GH treatment improves adult height in children with Noonan syndrome with and without mutations in protein tyrosine phosphatase, non-receptor-type 11 Eur. J. Endocrinol., September 1, 2008; 159(3): 203 - 208. [Abstract] [Full Text] [PDF]
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 A C Shaw, K Kalidas, A H Crosby, S Jeffery, and M A Patton The natural history of Noonan syndrome: a long-term follow-up study Arch. Dis. Child., February 1, 2007; 92(2): 128 - 132. [Abstract] [Full Text] [PDF]
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 J.-M. Limal, B. Parfait, S. Cabrol, D. Bonnet, B. Leheup, S. Lyonnet, M. Vidaud, and Y. Le Bouc Noonan Syndrome: Relationships between Genotype, Growth, and Growth Factors J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 300 - 306. [Abstract] [Full Text] [PDF]
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, Patients with mutations in PTPN11; 
, patients without mutations in PTPN11. n.s., Not significant.