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Department of Pediatrics, Keio University School of Medicine (R.Y., T.H.), Tokyo 160-8582, Japan; Division of Endocrinology and Metabolism, Tokyo Metropolitan Kiyose Children’s Hospital (Y.H.), Kiyose 204-8567, Japan; Department of Pediatrics, Dokkyo University School of Medicine Koshigaya Hospital (T.N.), Koshigaya 343-8555, Japan; Department of Pediatrics, Nagasaki University School of Medicine (E.K.), Nagasaki 852-8501, Japan; Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital (Y.T.), Ichikawa 252-8713, Japan; Department of Pediatrics, Toyama Medical and Pharmaceutical University (H.K.), Toyama 930-0194, Japan; Department of Pediatrics, Yamanashi University School of Medicine (K.O.), Kofu 409-3898, Japan; Department of Pediatrics, Tokyo Medical and Dental University School of Medicine (T.On.), Tokyo 113-8519, Japan; Hanew Endocrine Clinic (K.H), Sendai 980-0824, Japan; Divisions of Molecular Medicine (T.Ok.), Adolescent and Young Adult Medicine (R.H.), and Endocrinology and Metabolism (T.T.), National Center for Child Health and Development, Tokyo 154-8535, Japan; and Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development (R.Y., T.Og.), Tokyo 154-8567, Japan
Address all correspondence and requests for reprints to: Dr. Tsutomu Ogata, Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya, Tokyo 154-8567, Japan. E-mail: tomogata{at}nch.go.jp.
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Abstract |
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Introduction |
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In 2001, Tartaglia et al. (8) reported that NS is caused by heterozygous missense mutations of the gene for PTPN11 (protein-tyrosine phosphatase, nonreceptor type 11) on 12q24. The PTPN11 gene consists of 15 exons and encodes cytoplasmic tyrosine phosphatase with two tandemly arranged Src homology 2 (SH2) domains (N-SH2 and C-SH2) on the N-terminal side and a protein-tyrosine phosphatase (PTP) domain on the C-terminal side (8, 9, 10). PTPN11 is expressed in various tissues and plays a critical role in regulating the responses of eukaryotic cells to multiple extracellular signals, such as hormones, cytokines, and growth factors (9, 10, 11). According to the protein structural analysis, the N-SH2 domain binds the PTP domain in the absence of a tyrosine-phosphorylated peptide, inhibiting phosphatase activity, whereas the intermolecular interaction of the N-SH2 domain with a phosphopeptide dissociates intramolecular N-SH2/PTP binding, activating the phosphatase function (11). Because the missense mutations cause amino acid substitutions in and around the broad N-SH2/PTP interaction surface, they have been suggested to affect intramolecular N-SH2/PTP binding in the absence of a phosphopeptide, leading to excessive phosphatase activity (8). Indeed, such gain of function effects have been confirmed for representative missense mutants by functional studies (8, 12).
Thereafter, mutation analysis has been performed for a large number of NS patients, showing that PTPN11 mutations account for approximately 40% of patients with NS and result in not only common NS features, but also infrequent NS features, such as JMML, with considerable inter- and intrafamilial variations in the expressivity of the same mutations (12, 13, 14, 15, 16, 17, 18). Furthermore, phenotypic assessment has suggested that pulmonary valve stenosis (PS) is more frequent, and hypertrophic cardiomyopathy (HCM) is less prevalent in patients with mutations than in those without mutations, and that minor anomalies are grossly similar between mutation-positive and mutation-negative patients (13, 14, 15, 16, 17, 18).
In this study we report on PTPN11 mutations and clinical findings in 45 Japanese NS patients and discuss the correlations between genotypes and phenotypes, including the degree of growth deficiency and the occurrence of infrequent features that have been poorly examined to date.
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Subjects and Methods |
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This study consisted of 45 NS patients (25 males and 20 females), aged 0.1–34.5 yr (median, 15.8 yr), including previously reported patients (14, 19). All of the NS patients had the characteristic face with hypertelorism and met the diagnostic criteria proposed by van der Burgt et al. (20). Two NS patients were familial cases. One (a boy) was the proband of a two-generation family in which his mother also had the NS phenotype, and the other (a girl) was the proband of a three-generation family in which her grandmother, mother, aunt, and nephew also had the NS phenotype. The remaining 43 patients were sporadic cases. All of the patients had a normal karyotype.
Mutation analysis of PTPN11
After obtaining informed consent, the leukocyte genomic DNA of each patient was amplified by PCR for all 15 exons and the flanking introns of the PTPN11 gene. Subsequently, the PCR products were subjected to direct sequencing from both directions on a CEQ 8000 autosequencer (Beckman Coulter, Fullerton, CA; www.beckman.com). The primer sequences and the PCR conditions have been described previously (14). For controls, leukocyte genomic DNA from 100 normal subjects were similarly analyzed with permission.
Clinical assessment
Clinical assessment was performed for growth pattern, cardiovascular lesion, and other findings observed in NS. Furthermore, to evaluate the possible effect of ascertainment bias, the first clinical feature that led to the diagnosis of NS was examined in each case.
For the growth evaluation, birth length (BL) and birth weight (BW) were assessed by the gestational age-matched Japanese standards, and childhood (5–7 yr of age) height (CH) was evaluated using longitudinal growth standards for the Japanese (21). Target height (TH) was calculated in sporadic cases by equations for the Japanese (22) (TH was not calculated for familial cases, because it predicts the adult height of a child born to normal parents). To allow for comparison between different sexes and different ages, the growth data were expressed as an SD score (SDS).
For the cardiovascular assessment, auscultation, chest roentgenograms, electrocardiograms, and echocardiograms were obtained for all the patients. In addition, two-dimensional echocardiography, continuous wave Doppler flow analysis, and/or cardiac catheterization were also performed in patients with cardiovascular lesion to make a precise diagnosis.
Other findings were primarily evaluated clinically. Hematological abnormalities were based on biochemical data, and JMML was diagnosed on the basis of the World Health Organization’s guidelines (23).
Statistical analysis
After analyzing the normality of variables by the 
2 test and comparing the variances of two groups by the F test, the statistical significance of the mean was examined by t test, and that of the median was analyzed by Mann-Whitney’s U test. The statistical significance of the frequency was analyzed by Fisher’s exact probability test, and the correlation coefficient was determined by Pearson’s test. P < 0.05 was considered significant.
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Results |
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The results are shown in Fig. 1
. Heterozygous PTPN11 mutations were identified in 18 of the 45 NS patients (eight males and 10 females). Of the 18 patients, 17 were sporadic cases, and the remaining one was the proband of the two-generation family. The mutations consisted of a novel 3-bp deletion mutation and 10 recurrent missense mutations. They resided either in the N-SH2 or the PTP domain, and clustered at exon 3. They were absent in the 100 normal subjects.
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The results are summarized in Table 1. The age and sex ratio were similar in patients with PTPN11 mutations and those without PTPN11 mutations. Fifteen males and six females were first seen because of short to low-normal stature in childhood, 10 males and 13 females were recognized because of cardiac murmur at health checkups in infancy, and the remaining female was identified because of massive lymphedema during the fetal to neonatal period; thus, the ascertainment bias was detected for statural growth (15 of 25 vs. 6 of 20; P = 0.04), but not for cardiovascular lesion (10 of 25 vs. 13 of 20; P = 0.09).
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). CH-SDS and CH-SDS minus TH-SDS were also similar between mutation-positive and mutation-negative patients with cardiovascular lesion. There was no significant correlation between CH-SDS and TH-SDS in either mutation-positive or mutation-negative patients. Adult heights were available for only two mutation-positive patients (–2.3 and –4.6 SD) and for five mutation-negative patients (–1.8 to –4.9 SD; –3.4 ± 1.3 SD).
The growth pattern was further analyzed to evaluate the possible influence of the ascertainment bias for stature (Table 2). All of the growth parameters were comparable between mutation-positive males and females and between mutation-positive and mutation-negative males. However, CH-SDS minus TH-SDS, and CH-SDS and CH-SDS minus TH-SDS in patients with cardiovascular lesion were significantly different between mutation-negative males and females, as were CH-SDS, and CH-SDS and CH-SDS minus TH-SDS in patients with cardiovascular lesion, between mutation-positive and mutation-negative females.
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). Other cardiovascular anomalies included mitral valve insufficiency (MI) in a mutation-positive patient with S502T as well as MI, MI and ventral septal defect, MI and aortic valve stenosis, aortopulmonary fenestration, and teratology of Fallot in five mutation-negative patients. HCM was absent in mutation-positive patients and was present in five mutation-negative patients only, although the prevalence was not statistically significant. Electrocardiographic conduction abnormality was absent in all patients. In familial cases, PS was present in the proband with N308D and was absent in his mother with NS, and cardiovascular lesion was absent in the affected individuals of the three-generation family without the mutation.
Other features were grossly similar in the prevalence between the two groups (Table 1). However, hematological abnormalities were exclusively present in mutation-positive patients. Bleeding diathesis was due to thrombocytopenia, platelet dysfunction, and coagulation factor XII deficiency in three patients with N308D, D61G, and Q79R, respectively. JMML occurred in two patients with del60G and S502T during infancy and resolved spontaneously. In addition, penile hypospadias was exhibited by a boy with T73I who had bilateral inguinal testes approximately 1 ml in volume and no pubertal development at 15 yr of age. In this boy, a human chorionic gonadotropin test (3000 IU/m2·dose im for 3 consecutive days, with blood sampling on the first and the fourth days), yielded no testosterone response (0.4
0.4 ng/ml; 1.41.4 nmol/liter), and a GnRH test (100 µg/m2 bolus, iv; blood sampling at 0, 30, 60, 90, and 120 min) showed mild hyperresponses of LH (1.321.8 mIU/ml) and FSH (8.924.0 mIU/ml) compared with the Japanese reference data (24).
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Discussion |
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PTPN11 mutations were identified in 18 of the 45 NS patients (40%). The frequency in NS patients is reminiscent of that reported previously in Caucasian patients (
40%) (12, 15, 16, 17, 18). In addition, because the PTPN11 mutation was detected in one of the two familial cases, this provides further support for the previous finding that familial NS is also a genetically heterogeneous condition with and without PTPN11 mutations (13, 16, 18). Because most of the mutations are missense mutations residing in and around the broad N-SH2/PTP interaction surface, they would impair N-SH2/PTP binding, leading to excessive phosphatase activity (12, 13). In this context, the del60Gly mutation is noteworthy, because this is the sole deletion mutation identified for PTPN11. Because the 60th glycine residue of the N-SH2 domain is directly involved in the N-SH2/PTP interaction (11), loss of the glycine residue would also disrupt N-SH2/PTP binding, activating the phosphatase function. T42A mutation is also noteworthy, because the 42nd threonine residue lies apart from the N-SH2/PTP interaction surface (11). Because the 42nd threonine residue of the N-SH2 domain interacts directly with a tyrosine phosphate (11), T42A might gain a function to promote dissociation of N-SH2/PTP binding in the absence of a tyrosine phosphate. Alternatively, T42A might cause a conformational change in the N-SH2 domain, affecting the N-SH2/PTP interaction.
Several mutations are notable in terms of phenotypic features. First, Y62D and T73I identified in NS patients without JMML have been described in NS patients with JMML (12), and S502T detected in an NS patient with JMML has been reported in NS patients without JMML (15). This indicates the presence of plural mutations shared by NS patients with and without JMML. Second, Y279C found in one NS patient has been identified in one NS patient (13) and repeatedly identified in patients with LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormalities of genitalia, retardation of growth, sensorineural deafness) syndrome, an NS-related disorder (18, 25, 26). This represents the presence of a rare mutation shared by NS and LEOPARD syndrome.
Clinical features
Most patients, except for one female patient, were diagnosed because of growth problem or cardiovascular lesion. In this regard, short to low-normal stature in childhood was more frequently reported by males than by females, and this yielded the ascertainment bias for height in this study. It is likely that short stature is regarded as a more serious problem in males than in females. By contrast, cardiovascular lesion was similarly identified in males and females. This would be natural, because cardiovascular lesion was recognized by cardiac murmur at infantile checkups. Because more than half of the patients were first identified due to cardiac murmur, and cardiovascular anomalies were more frequent in mutation-positive patients than in mutation-negative patients, this would have contributed to the similar sex ratios of examined patients (25 males and 20 females) and of PTPN11 mutation-positive patients (8 males and 10 females) in the presence of the sex-related ascertainment bias for stature.
The growth patterns at birth and in childhood were comparable in mutation-positive and mutation-negative patients, although adult height was not analyzed. This implies that the degree of growth disadvantage is similar in patients with PTPN11 mutations and those with a hitherto unidentified mutant gene(s) for NS. Consistent with this, the frequency of short stature has been reported to be similar in mutation-positive and mutation-negative patients by Tartaglia et al. (13) and Maheshwari et al. (15), although it has been documented to be significantly higher in mutation-positive patients by Sarkozy et al. (18). In addition, our results are consistent with the classic auxological data showing that the average BL and BW remain within the normal range, and the average CH is just below –2 SD of the mean (27). Furthermore, the lack of a significant correlation between CH-SDS and TH-SDS is in contrast with the presence of a significant correlation between CH and midparental height in the normal population (28). This would primarily be due to the variable growth disadvantage in PTPN11 mutations.
Further growth analysis revealed that all of the growth parameters were comparable in mutation-positive males and females and in mutation-positive and mutation-negative males, whereas several parameters for childhood growth were significantly different in mutation-negative males and females and in mutation-positive and mutation-negative females. Collectively, these findings would imply that growth pattern is similar in a homogeneous group of mutation-positive patients regardless of sex and coincide with the sex-related ascertainment bias for stature.
Cardiovascular assessment showed that PS and ASD were more frequent in mutation-positive patients, whereas HCM was exclusively exhibited by mutation-negative patients. The results are grossly compatible with the previous data showing that PS is more prevalent in mutation-positive patients (13), and HCM is predominantly manifested by mutation-negative patients (13, 15, 16). Although the higher prevalence of ASD in mutation-positive patients has not been reported previously, it has been suggested that ASD tends to occur in patients with mutations at exon 3 (18). Thus, the high prevalence of ASD may be related to most mutations being identified at exon 3 in this study.
Several matters are noteworthy for the remaining features. First, hematological abnormalities were exclusively manifested by mutation-positive patients. This would be consistent with PTPN11 being involved in the signal transduction of hemopoietic cells (29). Second, in contrast to the poor prognosis of JMML in non-NS patients (30), JMML in the two PTPN11 mutation-positive patients occurred in infancy and resolved spontaneously. Consistent with this, PTPN11 mutations have been identified in NS patients with JMML, and spontaneous remission of JMML has been described previously (4, 5, 6, 7, 12, 31). This indicates the importance of PTPN11 analysis in NS patients with JMML, because spontaneous remission of JMML is expected in NS patients with proven PTPN11 mutations. In this context, because germline PTPN11 mutations have previously been identified in two patients with JMML who had short stature and PS, but lacked other characteristics of the NS phenotype (12), PTPN11 should also be examined in atypical NS patients with JMML. Third, hypospadias was present in a mutation-positive patient with endocrine data suggestive of primary hypogonadism. However, because the endocrine results at 15 yr of age would not necessarily indicate the endocrine status during fetal life, it is uncertain at present whether hypospadias formed in the fetal life is primarily due to testicular dysfunction or defective responsiveness of the external genital tissues to testosterone (32). Lastly, clinical features ascribed to lymphatic abnormalities and cleft palate were manifested by mutation-positive and mutation-negative patients. This implies that such features are caused by mutations of both PTPN11 and a hitherto unidentified gene(s) for NS.
In summary, despite the presence of NS-compatible clinical features in all cases examined, phenotypic assessment implies that a sex-related ascertainment bias exists for stature, and that the type of cardiovascular lesions and the prevalence of hematological abnormalities are different in mutation-positive and mutation-negative patients.
Clinical implications
Because NS has been shown to be a genetically heterogeneous condition in terms of PTPN11 mutations, this has several clinical implications. First, molecular confirmation of PTPN11 mutations should serve to establish the diagnosis of NS in patients with atypical features, although the lack of a PTPN11 mutation does not exclude the possibility of NS. Second, identification of PTPN11 mutations would permit appropriate genetic counseling. Third, some prognostic differences may be revealed between mutation-positive and mutation-negative patients by a long-term follow-up. Lastly, some differences may also be identified for the response to therapeutic intervention. In this regard, because PTPN11 has been shown to exert a negative regulatory effect on GH receptor signaling (33), growth failure in PTPN11 mutation-positive patients would more or less be ascribed to a decreased endogenous GH effect. Thus, it would be tempting to compare the effect of GH therapy in PTPN11 mutation-positive and mutation-negative patients. One may argue that GH therapy, especially that in PTPN11 mutation-positive patients, could increase the predisposition to malignancy, such as PTPN11-related myeloproliferative disorders. However, this possibility appears unlikely, because myeloproliferative disorders in NS are primarily caused by an activation of the RAS/mitogen-activated protein kinase cascade (12, 31) that is independent of GH receptor signaling. In addition, recent data argue against the malignant potential of GH therapy (34), and the occurrence of malignant diseases has not been reported in GH-treated NS patients (35).
Remarks and conclusions
Several points should be made with respect to the present study. First, the number of patients studied is still too small to make definite conclusions. Second, mutations with gain of function effects might exist in unexamined noncoding regions. Third, clinical features in NS are known to change with age (36), so that several features may have been unrecognized at the age of examination. For example, clinical features resulting from lymphatic hypoplasia/dysplasia tend to resolve with age (3), and skin pigmentation and HCM are usually discernible after infancy (37, 38). Thus, although it appears unlikely that some patients had subclinical chylothorax at an early age, it may be possible that some patients have skin pigmentation and/or HCM at a later age (this idea would be applicable to the patient with Y279C who was 6.5 yr of age at the time of investigation, because Y279C has frequently been identified in LEOPARD syndrome, which is characterized by diffuse skin pigmentation and is often associated with HCM) (18, 25, 26). Lastly, because assessment of somatic features is subjective, some features may be overlooked, and other features may be overestimated.
Despite the above caveats, the present study suggests that PTPN11 mutations account for approximately 40% of NS patients, as has been reported previously. Furthermore, clinical assessment implies that the type of cardiovascular lesions and probably the occurrence of hematological abnormalities as well are different in mutation-positive and mutation-negative patients, whereas the remaining findings are similar in the two groups of patients.
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Footnotes |
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Abbreviations: ASD, Atrial septal defect; BL, birth length; BW, birth weight; CH, childhood (5–7 yr of age) height; HCM, hypertrophic cardiomyopathy; JMML, juvenile myelomonocytic leukemia; MI, mitral valve insufficiency; NS, Noonan syndrome; PS, pulmonary valve stenosis; PTP, protein-tyrosine phosphatase; PTPN11, protein-tyrosine phosphatase, nonreceptor type 11; SDS, SD score; SH2, Src homology 2; TH, target height.
Received December 4, 2003.
Accepted March 28, 2004.
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