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PTPN11 (Protein-Tyrosine Phosphatase, Nonreceptor-Type 11) Mutations in Seven Japanese Patients with Noonan Syndrome

Department of Pediatrics (K.K., T.H., R.K., T.O.) and Pharmacia Fund for Growth & Development (T.S.), Keio University School of Medicine, Tokyo 160-8582, Japan; Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital (K.M.), Ichikawa 272-0853, Japan; Department of Pediatrics, Saitama Municipal Hospital (S.S.), Saitama 336-8522, Japan; National Children’s Hospital (N.M.), Tokyo 154-8509, Japan; Department of Pediatrics, Dokkyo University School of Medicine Koshigaya Hospital (T.N.), Koshigaya 343-0845, Japan; Division of Endocrinology and Metabolism, Tokyo Metropolitan Kiyose Children’s Hospital (Y.H.), Kiyose 204-8567, Japan; and Department of Pediatrics, Tokyo Electric Power Company Hospital (T.O.), Tokyo 160-0016, Japan

Address all correspondence and requests for reprints to: Dr. Tsutomu Ogata, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: . t-ogata{at}po.iijnet.or.jp

Abstract

Noonan syndrome is an autosomal dominant disorder defined by short stature, delayed puberty, and characteristic dysmorphic features. Tartaglia et al. (Nature Genetics, 29:465–468) have recently shown that gain-of-function mutations in the gene PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) cause Noonan syndrome in roughly half of patients that they examined. To further explore the relevance of PTPN11 mutations to the pathogenesis of Noonan syndrome, we analyzed the PTPN11 gene in 21 Japanese patients. Mutation analysis of the 15 coding exons and their flanking introns by denaturing HPLC and direct sequencing revealed six different heterozygous missense mutations (Asp61Gly, Tyr63Cys, Ala72Ser, Thr73Ile, Phe285Ser, and Asn308Asp) in seven cases (six sporadic and one familial). The mutations clustered either in the N-Src homology 2 domain or in the protein-tyrosine phosphatase domain. The clinical features of the mutation-positive and mutation-negative patients were comparable. The results provide further support to the notion that PTPN11 mutations are responsible for the development of Noonan syndrome in a substantial fraction of patients and that relatively infrequent features of Noonan syndrome, such as sensory deafness and bleeding diathesis, can also result from mutations of PTPN11.

NOONAN SYNDROME IS an autosomal dominant disorder characterized by proportionate short stature, delayed puberty, cardiac anomalies, and multiple minor anomalies, such as hypertelorism, downward slanting palpebral fissures, malrotated ears, webbed neck, low posterior hairline, and cubitus valgus (1, 2, 3). Mild mental retardation, hearing difficulty, and bleeding diathesis are also occasionally observed in affected individuals, as are hypoplastic external genitalia and cryptorchidism in affected males. This condition is relatively common, with an estimated incidence of 1 in 1000–2500 live births (1).

A gene for Noonan syndrome has been mapped to 12q24. Jamieson et al. (4) and Brady et al. (5) assigned the gene to an approximately 14-centimorgan (cM) region between D12S105 and NOS1 based on a linkage analysis of a three-generation Dutch family, and Legius et al. (6) localized the gene to an approximately 5-cM region between D12S84 and D12S1341 by an analysis of a four-generation Belgian family. These results imply that a gene for Noonan syndrome resides in an approximately 4-cM region from D12S105 to D12S1341, which overlaps the critical regions defined by the two linkage studies.

Tartaglia et al. (7) recently identified nine different heterozygous missense mutations in the PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) gene in 13 of 24 patients with sporadic or familial form of Noonan syndrome. 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). PTPN11 is widely expressed in human tissues, especially in the heart, brain, and skeletal muscle (8, 9), and plays a critical role in regulating the responses of eukaryotic cells to extracellular signals (10). Tartaglia et al. (7) have also indicated, on the basis of an energetics-based structural analysis of two N-SH2 mutants, that the missense mutations identified in patients with Noonan syndrome have gain-of-function effects leading to excessive PTPN11 activity (7).

However, because the clinical features of the patients with PTPN11 mutations were not documented in the original report by Tartaglia et al. (7), and there have been no other reports describing mutations of the PTPN11 gene, we screened Japanese patients with Noonan syndrome for PTPN11 mutations and summarized clinical features of mutation-positive cases.

Materials and Methods

Patients

The study protocol was approved by the Ethics Committee of Keio University School of Medicine. After obtaining informed consent, 21 Japanese patients (9 males and 12 females) with Noonan syndrome were screened for mutations of the PTPN11 gene. All of the patients had characteristic minor anomalies, and ten of them had cardiac diseases such as pulmonary valve stenosis or cardiomyopathy. Nineteen cases were sporadic, and the other two were familial cases from a two-generation family and a three-generation family, respectively. As controls, 100 clinically normal Japanese subjects were similarly analyzed with their permission. All of the Noonan syndrome patients and control subjects had a normal karyotype.

Mutation analysis of the PTPN11 gene

Using the cDNA sequence of the human PTPN11 gene (GenBank accession no. NM002834) as the query, a homology search was performed with the BLAST2 program (http://www.ncbi.nlm.nih.gov/blast2.html) against the GenBank database. The genomic sequences (GenBank accession nos. AC004086 and AC004216 were found to be highly homologous to PTPN11 cDNA sequences. Based on the genomic structure delineated, PCR primers were designed to cover the complete coding sequence with flanking intronic sequences, as described previously (11).

Leukocyte genomic DNA was amplified for the 15 exons and flanking introns of the PTPN11 gene by PCR with 15 sets of primers (Table 1). PCR amplification was performed as described previously (12). Mutation analysis was performed using two complementary approaches: exon 3 and exon 8, which have previously been shown to be mutation hot spots (7), were screened by direct sequencing of the PCR products. The PCR products were sequenced from both directions on an ABI3100 autosequencer (ABI, Foster City, CA). The remaining exons were screened by the denaturing HPLC (DHPLC) (WAVE, Transgenomic, San Jose, CA). This method, a variation of the heteroduplex assay, possesses high (>95%) sensitivity and specificity (13, 14). When abnormal chromatograms were detected, corresponding PCR products were directly sequenced. The optimal condition of each exon for DHPLC analysis was predicted by WAVE Maker software version 4.1.

Birth length and weight were assessed based on the gestational age-matched Japanese standards, and postnatal height/length and weight were evaluated by the longitudinal growth standards for the Japanese (15). Body proportion (the ratio of sitting height to leg length) was assessed by the age-matched standards for Japanese (16). Target height and target range were calculated by a modified Tanner’s equation for the Japanese with a positive height secular trend (17) for sporadic cases (target height was not calculated for familial cases, because it predicts the adult height/range of a child born to normal parents). Pubertal stage (genitalia in males, breast in females, and pubic hair in both sexes) was evaluated by the classification of Tanner (18), and menarchial age was assessed by the Japanese female standard (menarchial age in Japanese girls: 12.25 ± 1.25 yr) (19). Dysmorphic features were evaluated clinically, and cardiac anomalies were diagnosed based on either the echocardiography or cardiac catheterization findings.

Results

Mutation analysis of the PTPN11 gene

Six different PTPN11 mutations were identified in seven patients with Noonan syndrome (Fig. 1 and Table 2). All of the cases but one were sporadic. The familial case represented a mother-son pair, but the affected mother declined molecular testing. All six mutations were heterozygous missense mutations, and they clustered in the exon 3 encoding the N-SH2 domain or in the exon 8 encoding the PTP domain. None of these mutations were present in 100 normal individuals (i.e. 200 normal chromosomes).

View larger version (24K): [in this window] [in a new window]   Figure 1. Sequencing chromatogram of the novel PTPN11 mutations in patients with Noonan syndrome. Top, Case5 with Thr73Ile substitution. Bottom, Case 6 with Phe285Ser substitution.

The clinical features of the patients with the PTPN11 mutations are summarized in Table 3. Birth size was below the mean in most cases, with intrauterine growth retardation in case 5 and exceptionally large size in case 3. Length was low-normal in case 2 in infancy, and height was below -2 SD of the mean in cases 1 and 3–7, with severe short stature in cases 1, 5, and 6 in their middle to late teens. Body proportion was within the normal range in cases 1, 4, and 5. Pubertal development was impaired to various degrees in cases 1 and 3–6. Development was delayed in cases 2 and 4. Dysmorphic features were present with variable severity in cases 1–7, and cardiac anomalies were identified in cases 3–7. Sensory deafness was present in case 5. In addition, case 1 was afflicted with a bleeding diathesis manifested by recurrent sc bleeding and severe bleeding at the time of dental extraction. Laboratory tests for the bleeding diathesis showed abnormal bleeding time (16 min) (normal range, 4–8 min), borderline to low platelet count (131,000/µl) (150,000–350,000/µl), normal activated partial thromboplastin time (32.1 sec) (23–36 sec), and prothrombin time (12.1 sec) (10–13 sec). Platelet aggregations were mildly defective in response to ADP, collagen, epinephrine, and ristocetin (detailed charts not shown), low platelet retention rate by the Hellem II method (25%) (45–90%). The mother of case 7 had typical features of Noonan syndrome (Table 3).

Discussion

The present study revealed 6 PTPN11 mutations in 7 of 21 patients with Noonan syndrome. Two of the six mutations are novel, and four (7) had been identified previously. These findings provide further evidence for PTPN11 mutations being responsible for the development of Noonan syndrome. All of the PTPN11 mutations detected to date have been heterozygous single base substitutions and have been clustered almost exclusively either in exon 3 or exon 8 (Fig. 2). All the base changes resulted in amino acid substitutions within the subregions of the N-SH2 and PTP domains where the two domains interact to negatively regulate the phosphatase activity of the protein (10, 20). Hence, missense mutations in these subregions would disrupt the intramolecular interaction between the N-SH2 and the PTP domain (7), leading to a conformational change into a constitutively active form.

View larger version (18K): [in this window] [in a new window]   Figure 2. Distribution of the 11 different mutations detected to date. The six different mutations accompanied by asterisks were identified in the present study, and the others were detected by Tartaglia et al. (7 ). The numbers of cases with each mutation are indicated in parentheses.

Less common features among the PTPN11 mutation-positive cases included developmental delay, bleeding diathesis, and sensory deafness. Hence, the wide phenotypic spectrum appreciated since the early descriptions of Noonan syndrome (2, 3) was also demonstrated among the PTPN11 mutation-positive cases. The observed pleiotropic effects of PTPN11 mutations most likely reflect the diverse role of PTPN11 in regulating multiple signal transduction pathways involving growth factors (e.g. epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor), cytokines, and hormones (e.g. GH) (10).

No distinctive genotype-phenotype correlation, however, was elucidated among the PTPN11 mutation-positive cases, with variable expression of the same mutation instead being evident both in an interfamilial and intrafamilial context. The mutation Tyr63Cys leads to cardiac defect (atrial septal defect) in case 3 but none in case 2. Similarly, in the familial case the child with the Asn308Asp mutation had pulmonary stenosis, but his mother had no cardiac defects. It would appear difficult to predict phenotypic outcome from the genotype. The genotype of an as yet unknown modifying locus may influence the phenotype of patients with PTPN11 mutations. In support of this, modification of the phenotype of the epidermal growth factor receptor mutant by mutations in the Ptpn11 gene has been observed, at least in a mouse model (21).

In the present study, PTPN11 mutations were not identified in a significant portion of the patients (14 out of 21) who had classic features of Noonan syndrome. Phenotypic features of PTPN11 mutation-negative cases were comparable to those of the mutation-positive cases. The relatively high frequency of PTPN11 mutation-negative cases, which was documented by Tartaglia et al. (7) as well (11 out of 24), may point to genetic heterogeneity of Noonan syndrome. Indeed, the locus heterogeneity of Noonan syndrome has been indicated by a linkage analysis of a family of Noonan syndrome who did not show linkage to the 12q24 critical region (4). Clinically well-documented cases of Noonan syndrome who were born to consanguineous parents (22, 23) also support the existence of an autosomal recessive, rather than autosomal dominant, form of Noonan syndrome. Furthermore, documentation of the Noonan syndrome phenotype in patients with t(5;7)(p15;q22) (24), t(3;22)(p21;q13), del(3)(q11q21) (25), del(12)(q12q13.12) (26), del(13)(q21.32q22.3) (27), and dup(11)(q13.3q14.2) (28) may suggest the presence of additional loci responsible for the pathogenesis of Noonan syndrome.

In summary, this study lends further support for the notion that PTPN11 mutations cause Noonan syndrome. Molecular analysis of the PTPN11-related signal transduction processes in conjunction with clinical observations such as lymphatic dysplasia (29) will serve to clarify the pathogenesis of Noonan syndrome.

Acknowledgments

Footnotes

This work was supported in part by grants from the Ministry of Health and Welfare, Sankyo Co., Ltd. Foundation of Life Science, the Japan Health Science Foundation, and Pharmacia Fund for Growth & Development Research.

Abbreviations: DHPLC, Denaturing HPLC; PTP, protein-tyrosine phosphatase; PTPN11, protein-tyrosine phosphatase, nonreceptor-type 11; SH2, Src homology 2.

Received February 5, 2002.

Accepted April 8, 2002.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kosaki, K. Articles by Ogata, T. Search for Related Content PubMed PubMed Citation Articles by Kosaki, K. Articles by Ogata, T.