2019 Volume 66 Issue 11 Pages 983-994
Noonan syndrome (NS) is a heterogeneous disorder with multiple congenital malformations. Recent advances in molecular and genetic approaches have identified a number of responsible genes for NS, most of which are components of the RAS/MAPK signaling pathway, and genotype-phenotype correlation analyses have been extensively performed; however, analysis of Japanese NS patients is limited. Here, we evaluated clinical characteristics in genetically diagnosed NS patients and their relationships to genotypes. A total of 48 clinically diagnosed NS were included, and responsible mutations were identified in 39 patients (81.3%) with PTPN11 mutations being the most prevalent followed by SOS1 mutations. Cardiac anomalies including pulmonary stenosis and hypertrophic cardiomyopathy were most prevalent (87.2%), and the prevalence of hypertrophic cardiomyopathy was greater in patients without PTPN11 mutations than in those with PTPN11 mutations. Short stature was the second-most prevalent (69.2%) characteristic, and present height SD score was significantly associated with height SD score at 1 year old. Patients with SOS1 mutations had greater present height SD score and better growth during infancy. These findings suggest the presence of a genotype-phenotype correlation in Japanese patients with NS, which enables us to use genetic information to predict the clinical course and may allow for genotype-based medical interventions.
NOONAN SYNDROME (NS, OMIM #163950), first described by Jacqueline Noonan [1], is a clinically heterogeneous disorder with multiple congenital malformations, and clinical characteristics of NS include dysmorphic facial features, short stature, congenital heart diseases, and parenchymal abnormalities and commodities [2]. NS is an autosomal dominant disorder with a prevalence of 1 in 1,000–2,500 live births [3], but the prevalence in Japan has been assumed to be 1 in 10,000 live births (the RAS/MAPK syndromes homepage http://www.medgen.med.tohoku.ac.jp/RasMapk%20syndromes.html). The diagnosis of NS is usually based on clinical findings using the scoring system by van der Burgt [4], but dysmorphic facial characteristics become subtler at adulthood, which makes clinical diagnosis more difficult. Since the discovery of PTPN11 (protein-tyrosine phosphatase, nonreceptor type 11) as a responsible gene for NS [5], numerous genes, including SOS1 [6], RAF1 [7, 8], KRAS [9], NRAS [10], BRAF [11] and RIT1 [12], components of the RAS (Ras-mitogen-activated protein kinase)/MAPK signaling pathway, have been identified for the diagnosis of NS. Because mutations in genes related to the RAS/MAPK signaling pathway have also been identified in clinically overlapping NS-related disorders, such as Costello syndrome and CFC (cardio-facio-cutaneous) syndrome, the term RASopathies is used to represent disorders caused by abnormalities in this signaling pathway [13-15].
The establishment of a molecular basis of NS has made it possible to use genetic analysis to diagnose NS, and genotype-phenotype correlation analyses have been extensively performed to understand genotype-specific features of NS. For example, NS patients with mutations in the PTPN11 gene are shorter than those without PTPN11 mutations [16], and this is associated with decreases in IGF1 levels [17]; however, there is also evidence showing a lack of this association [18]. In addition, NS patients with SOS1 mutations are taller, whereas patients with SHOC2 mutations are shorter in a Brazilian population [19]. Pulmonary stenosis is more prevalent in PTPN11 mutation-positive patients [18], and hypertrophic cardiomyopathy is more frequently observed in patients with RAF1 mutations [7]. Although these findings enable us to use genetic information to predict the clinical course of NS and may allow for genotype-based medical interventions, genotype-phenotype analysis has not been extensively performed in Japanese NS patients. To provide more evidence for genotype-phenotype correlations in Japanese NS patients, we examined the clinical features of Japanese patients who were diagnosed with NS by a genetic test and analyzed genotype-phenotype associations.
We retrospectively evaluated the medical records of 48 patients clinically diagnosed with Noonan syndrome (M: 26, F: 22) before 31 May 2017 at Osaka Women’s and Children’s Hospital. The clinical characteristics of patients are shown in Table 1. Clinical diagnosis was made based on the Van der Burgt clinical scoring system by a medical geneticist [20]. When the patient had a facial feature unique to NS and one additional clinical characteristic including cardiac anomaly, short stature, pectus deformity, mental retardation, cryptorchidism, or family history of NS, the patient was diagnosed as having NS. When a facial feature was less characteristic to NS, two additional clinical features were required. Genetic testing was performed in all subjects. This study was approved by the Ethical Committee of Osaka Women’s and Children’s Hospital (approval No. 1138).
| No. | Gene mutation | cDNA | Protein | Sex | Age | Height SDS (SD) | Target height (SD) | GA (week) | Weight SDS at birth (SD) | Height SDS at birth (SD) | Heart disease (cardiac surgery and age) | ECG |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | PTPN11 | c.923A>C | p.N308T | M | 1y 0m | –6.4 | NA | 36 | 2.6 | NA | HCM, VSD, ASD | NA |
| 2 | PTPN11 | c.184T>G | p.Y62D | F | 2y 4m | –3.5 | 0.4 | 38 | –0.6 | 0.5 | ASD (direct closure at 11yo), PS | RSR’, inverted T wave (V4) |
| 3 | PTPN11 | c.1510A>G | p.M504V | M | 3y 1m | –2.3 | 0.0 | 37 | 1.5 | 1.0 | PS | borderline Q on V5,V6 |
| 4 | PTPN11 | c.854T>C | p.F285C | F | 4y 1m | –2.2 | NA | 38 | 1.6 | –0.8 | ASD, PS | normal |
| 5 | PTPN11 | c.922A>G | p.N308D | M | 4y 7m | –3.1 | –0.3 | 38 | –0.4 | –0.8 | PS | normal |
| 6 | PTPN11 | c.922A>G | p,N308D | M | 6y 1m | –2.1 | –1.0 | 38 | –0.9 | –1.5 | PS | normal |
| 7 | PTPN11 | c.317A>C | p.D106A | M | 7y 3m | –2.7 | 0.2 | 38 | –0.3 | –0.1 | (–) | normal |
| 8 | PTPN11 | c.1471C>G | p.P491A | M | 7y 4m | –2.7 | 0.8 | 40 | 1.7 | 1.7 | PS, ASD | right ventricular hypertrophy (mild), clock roatation |
| 9 | PTPN11 | c.218C>T | p.T73I | M | 9y 5m | –5.7 | NA | 32 | 0.0 | –1.8 | (–) | normal |
| 10 | PTPN11 | c.922A>G | p.N308D | F | 10y 0m | –1.9 | 0.5 | 40 | 0.2 | –0.1 | VSD, HCM | abnormal Q (I,V5,V6), high T (V3,V4) |
| 11 | PTPN11 | c.922A>G | p.N308D | M | 11y 5m | –2.8 | 0.2 | 39 | –0.5 | 0.5 | PS | right axis deviation |
| 12 | PTPN11 | c.836A>G | p.Y279C | F | 11y 7m | 0.1 | NA | 40 | 2.6 | 0.9 | VSD | NA |
| 13 | PTPN11 | c.922A>G | p.N308D | F | 12y 10m | –3.3 | –0.6 | 40 | –0.4 | 0.4 | ASD | NA |
| 14 | PTPN11 | c.922A>G | p.N308D | M | 13y 1m | –2.5 | NA | 41 | 0.7 | 0.6 | ASD, PS | NA |
| 15 | PTPN11 | c.188A>G | p.Y63C | M | 14y 8m | –4.6 | –1.6 | 41 | –1.2 | –2.7 | PFO | normal |
| 16 | PTPN11 | c.844A>G | p.I282V | M | 20y 3m | –1.5 | –0.7 | 39 | –0.3 | –0.4 | PS | NA |
| 17 | PTPN11 | c.922A>G | p.N308D | F | 20y 7m | –2.0 | –0.9 | 37 | 0.3 | NA | ASD, PS | RSR’, clock roatation, left axix deviation |
| 18 | RAF1 | c.786T>G | p.N262K | F | 5y 2m | –1.5 | 0.3 | 40 | –0.2 | –0.3 | HCM | WPW, prolonged QT, clock rotation, elevated ST (V1,V2,V3) |
| 19 | RAF1 | c.776C>T | p.S259F | F | 8y 5m | –5.0 | 0.0 | 38 | 1.5 | –0.5 | HCM, PS, MR, LVOPO (release of stenosis at 11yo) | supraventricular tachycardia |
| 20 | RAF1 | c.768G>T | p.R256S | M | 14y 0m | –2.1 | 0.2 | 40 | 0.5 | –1.0 | HCM | flat II, RSR’ |
| 21 | RAF1 | c.770C>T | p.S257L | M | 14y 4m | –4.4 | 0.3 | 39 | 1.4 | –0.5 | HCM, LVOTO, AS, MR | abnormal Q (III,aV), prolonged QT, elevated ST (V1,V2), clock roatation, left axis deviation |
| 22 | RAF1 | c.1837C>G | p.L613V | M | 18y 2m | –2.1 | NA | 29 | 1.4 | NA | HCM | normal |
| 23 | RAF1 | c.770C>T | p.S257L | M | 22y 11m | –2.7 | NA | 40 | NA | NA | HCM | supraventricular tachycardia |
| 24 | SOS1 | c.1322G>A | p.C441Y | M | 1y 3m | 0.3 | NA | 39 | 0.2 | 0.3 | PS, HCM | anticlock roataion, inverted T wave |
| 25 | SOS1 | c.508A>G | p.K170E | F | 2y 7m | –0.7 | –1.0 | 39 | 0.7 | 0.6 | VSD | NA |
| 26 | SOS1 | c.1655G>A | p.R552K | F | 3y 9m | –1.6 | NA | 38 | 1.5 | –1.0 | PS, ASD | right axis deviation |
| 27 | SOS1 | c.1654A>G | p.R552G | M | 6y 7m | –1.2 | NA | 37 | 2.0 | 0.1 | (–) | NA |
| 28 | SOS1 | c.806T>G | p.M269R | M | 7y 4m | –1.5 | NA | 40 | 1.7 | 0.8 | PS | NA |
| 29 | SOS1 | c.797C>A | p.T266K | F | 11y 11m | –3.2 | –0.8 | 35 | 2.7 | 0.4 | PS, DORV (release of stenosis at 1yo) | right axis deviation |
| 30 | SOS1 | c.1297G>A | p.E433K | M | 13y 8m | –2.1 | NA | 38 | –0.3 | –1.4 | (–) | NA |
| 31 | KRAS | c.458A>T | p.D153V | F | 4y 1m | –3.2 | 0.3 | 36 | 1.9 | 0.2 | PS | NA |
| 32 | KRAS | c.178G>C | p.G60R | F | 6y 2m | –4.3 | NA | 31 | 0.4 | –0.9 | HCM, PS | NA |
| 33 | KRAS | c.458A>T | p.D153V | F | 21y 5m | –2.5 | NA | 35 | 2.0 | 0.4 | HCM | left axis deviation |
| 34 | KRAS | c.458A>T | p.D153V | M | 28y 8m | –4.4 | NA | 37 | –0.2 | 2.4 | (–) | NA |
| 35 | KRAS | c.458A>T | p.D153V | F | 39y 7m | –4.8 | NA | 38 | –1.0 | –0.3 | ASD, PS, PH | NA |
| 36 | BRAF | c.1408A>C | p.T470P | F | 1y 10m | –0.9 | NA | 37 | 1.1 | 0.8 | PS | NA |
| 37 | BRAF | c.1455G>C | p.L485F | F | 12y 8m | –2.5 | 0.1 | 36 | 0.7 | 0.2 | ASD, PS, HCM | normal |
| 38 | RIT1 | c.170C>G | p.A57G | F | 6y 0m | 0.9 | 2.2 | 36 | 2.5 | –0.2 | PS, HCM | NA |
| 39 | RIT1 | c.170C>G | p.A57G | F | 24y 11m | 0.7 | –0.4 | 37 | 2.0 | 1.4 | ASD (patch closure at unknown age), PS, HCM | small Q (II, III, aVF), T inversion |
| 40 | negative | M | 7m | –1.1 | NA | 32 | 2.8 | 0.1 | PFO | NA | ||
| 41 | negative | M | 2y 1m | –2.1 | NA | 31 | 1.7 | –0.2 | ASD | normal | ||
| 42 | negative | F | 3y 6m | –3.4 | –0.4 | 39 | –0.2 | –0.8 | ASD, PS | left axis deviation | ||
| 43 | negative | F | 4y 2m | –3.0 | –0.3 | 36 | –0.1 | –1.0 | PS, ASD (direct closure at 1yo), HCM | prolnged QT, abnormal ST-T (II, V4, V5) | ||
| 44 | negative | F | 7y 8m | –1.7 | NA | 37 | 0.8 | –0.4 | PS, ASD, HCM,VSD | NA | ||
| 45 | negative | M | 8y 6m | –3.1 | NA | 40 | –3.1 | –1.4 | (–) | normal | ||
| 46 | negative | M | 11y 8m | –0.6 | –0.4 | NA | 1.2 | 0.4 | PS | normal | ||
| 47 | negative | M | 16y 1m | –3.7 | NA | 31 | –0.4 | –0.5 | (–) | NA | ||
| 48 | negative | M | 16y 5m | –2.3 | NA | 40 | –2.3 | –0.9 | ASD, PS | NA |
PFO, patent foramen ovale; HCM, hypertrophic cardiomyopathy; (–), not observed; ASD, atrial septal defect; LVOTO, left ventricular outflow tract obstraction; NA, not available; PS, pulmonary stenosis; MR, mitral regurgitation; VSD, ventricular septal defect; DORV, double-outlet right ventricle
| No. | Gene mutation | Protein | Sex | Pectus deformity | Developmental disorder | Urological disease | Neurologocal disease | Otolaryngeal disease (severerity) | Lymphatic malformation |
|---|---|---|---|---|---|---|---|---|---|
| 1 | PTPN11 | p.N308T | M | (–) | MR | (–) | epilepsy | (–) | hydrops |
| 2 | PTPN11 | p.Y62D | F | (–) | NA | (–) | (–) | sensorineural hearing loss (moderate) | posterior cervical edema |
| 3 | PTPN11 | p.M504V | M | shield-shaped chest | MR | cryptorchidism | (–) | sensorineural hearing loss (profound) | (–) |
| 4 | PTPN11 | p.F285C | F | (–) | MR | (–) | (–) | (–) | (–) |
| 5 | PTPN11 | p.N308D | M | shield-shaped chest | MR | hydrocele testis | craniosynostosis | (–) | (–) |
| 6 | PTPN11 | p.N308D | M | pectus excavatum | MR, ADHD | cryptorchidism | (–) | (–) | (–) |
| 7 | PTPN11 | p.D106A | M | NA | MR | cryptorchidism | (–) | (–) | (–) |
| 8 | PTPN11 | p.P491A | M | pectus carinatum | MR, ADHD | cryptorchidism | craniosynostosis, epilepsy | sensorineural hearing loss (severe) | (–) |
| 9 | PTPN11 | p.T73I | M | NA | MR | cryptorchidism | (–) | sensorineural hearing loss (profound) | hydrops |
| 10 | PTPN11 | p.N308D | F | pectus excavatum | MR | (–) | craniosynostosis | (–) | (–) |
| 11 | PTPN11 | p.N308D | M | (–) | MR, ADHD | bilateral hydronephrosis | (–) | (–) | (–) |
| 12 | PTPN11 | p.Y279C | F | (–) | MR | (–) | epilepsy | sensorineural hearing loss (unknown) | (–) |
| 13 | PTPN11 | p.N308D | F | NA | ADHD, LD | (–) | (–) | (–) | (–) |
| 14 | PTPN11 | p.N308D | M | (–) | ADHD, MR | cryptorchidism | (–) | (–) | (–) |
| 15 | PTPN11 | p.Y63C | M | NA | LD | cryptorchidism | (–) | (–) | (–) |
| 16 | PTPN11 | p.I282V | M | barrel-shaped chest | ADHD, ASD | (–) | (–) | (–) | (–) |
| 17 | PTPN11 | p.N308D | F | NA | (–) | (–) | (–) | (–) | lymphangioma |
| 18 | RAF1 | p.N262K | F | (–) | (–) | (–) | (–) | enlarged tonsils | (–) |
| 19 | RAF1 | p.S259F | F | barrel-shaped chest | MR | (–) | hydrencephalus | conductive hearing loss (mild) | (–) |
| 20 | RAF1 | p.R256S | M | NA | (–) | (–) | (–) | (–) | (–) |
| 21 | RAF1 | p.S257L | M | NA | MR, ADHD | bladder tumor (at 4yo) | (–) | enlarged tonsils | (–) |
| 22 | RAF1 | p.L613V | M | (–) | (–) | (–) | epilepsy | (–) | hydrops |
| 23 | RAF1 | p.S257L | M | pectus carinatum | NA | NA | NA | NA | hydramnion |
| 24 | SOS1 | p.C441Y | M | (–) | MR | (–) | (–) | sensorinural hearing loss (severe) | hydrops |
| 25 | SOS1 | p.K170E | F | (–) | MR | (–) | s/o craniosynostosis | (–) | hydramnion, posterior cervical edema |
| 26 | SOS1 | p.R552K | F | (–) | MR | (–) | (–) | (–) | (–) |
| 27 | SOS1 | p.R552G | M | NA | ADHD, LD | (–) | (–) | enlarged tonsils, conductive hearing loss (severe) | hydrops |
| 28 | SOS1 | p.M269R | M | NA | NA | cryptorchidism | (–) | otitis | (–) |
| 29 | SOS1 | p.T266K | F | (–) | MR | (–) | corpus callosum agenesis, epilepsy | (–) | hydrops |
| 30 | SOS1 | p.E433K | M | NA | MR, ADHD | (–) | (–) | (–) | (–) |
| 31 | KRAS | p.D153V | F | (–) | MR | (–) | epilepsy | sensorineural hearing loss (unknown) | hydrops |
| 32 | KRAS | p.G60R | F | (–) | (–) | (–) | craniosynostosis | (–) | fetal pleural effusion |
| 33 | KRAS | p.D153V | F | NA | ADHD | (–) | (–) | (–) | (–) |
| 34 | KRAS | p.D153V | M | NA | psychiatric disease | (–) | (–) | (–) | NA |
| 35 | KRAS | p.D153V | F | NA | MR | (–) | NA | NA | NA |
| 36 | BRAF | p.T470P | F | (–) | MR | (–) | (–) | NA | NA |
| 37 | BRAF | p.L485F | F | NA | MR | (–) | s/o callosum agenesis | dysarthria | (–) |
| 38 | RIT1 | p.A57G | F | NA | (–) | (–) | (–) | (–) | (–) |
| 39 | RIT1 | p.A57G | F | (–) | MR | (–) | (–) | NA | PLE |
| 40 | negative | M | pectus excavatum | MR | hydrocele testis | (–) | (–) | hydrops | |
| 41 | negative | M | (–) | NA | cryptorchidism | (–) | (–) | hydrops | |
| 42 | negative | F | (–) | (–) | NA | (–) | (–) | (–) | |
| 43 | negative | F | NA | (–) | (–) | (–) | (–) | (–) | |
| 44 | negative | F | (–) | MR | (–) | (–) | (–) | (–) | |
| 45 | negative | M | NA | MR | cryptorchidism | (–) | (–) | hydrops | |
| 46 | negative | M | shortening sternum | MR | (–) | (–) | (–) | hydrops | |
| 47 | negative | M | NA | MR | hydrocele testis | (–) | hearing loss (mild, type: unknown) | (–) | |
| 48 | negative | M | NA | MR | cryptorchidism | (–) | (–) | (–) |
(–), not observed; LD, learning disability; PLE, protein-loosing eneteropathy; NA, not available; ADHD, attention-deficit hyperactivity disorder; MR, mental retardation
Clinical characteristics, including cardiovascular anomalies, developmental disorders, pectus deformities, urological complications, and otolaryngeal diseases, and responsible mutations, were retrospectively assessed based on medical records. Anthropometric measurements including standing height and weight with lightweight clothes, were measured using a digital scale (TANITA DC-250), and height SD score (SDS) was calculated based on normal growth standards for Japanese children based on the year 2000 national survey data [21], and height SDS below –2.0 for age was defined as having short stature. Birth length and weight were assessed based on the gestational age-matched Japanese standards. All the subject were followed by medical geneticists and NS-associated complications were evaluated by consulting to the specialized doctors or by performing detailed interview when NS-associated complications were evaluated in other hospitals. When cardiac complications were evaluated in our hospital, chest x-rays, electrocardiograms, and echocardiograms were performed by pediatric cardiologists. Other clinical features were evaluated by medical specialists including pediatric neurologists, pediatric endocrinologists, urologists, and otolaryngologists. Hearing loss was diagnosed using audiogram and its severity was classified based on configuration of the audiogram as follows: mild; 25–40 dB, moderate; 40–70 dB, severe; 70–90 dB, and profound; 90–120 dB. IGF-1 levels were determined by an immunoradiometric assay using somatomedin CIISIEMENS before March 2012 and IGF-1 IRMA DAIICHI after April 2012. IGF-1 SDS was calculated based on the Japanese reference values [22]
Mutational analysisGenomic DNA was purified from whole blood of the subjects. Genetic analysis of their parents to confirm whether the identified mutations were inherited or de novo mutations was not performed. Exons 1–15 in PTPN11 (Protein tyrosine phosphatase, non-receptor type 11) gene, all the exons in SOS1 (SOS Ras/Rac guanine nucleotide exchange factor-1) gene, exons 7, 14, and 17 in RAF1 (Raf-1 proto-oncogene, serine/threonine kinase) gene, exons 1–5 in KRAS (KRAS proto-oncogene, GTPase) gene, exons 6 and 11–16 in BRAF (B-Raf proto-oncogene, serine/threonine kinase) gene, exon 1 in SHOC2 gene, and all exons in RIT1 (Ras-like without CAAX1) gene were sequenced based on Sanger methods. The primers used are available upon request.
Statistical analysisAll data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with the Student’s t-test or ANOVA. Correlation between two variables was statistically analyzed by Pearson’s correlation analysis. Genotype-phenotype correlations were analyzed using a Chi-square test. P-values less than 0.05 were considered significant.
Genetic testing was performed in a total of 48 patients clinically diagnosed as having NS, which revealed that 39 patients (81.3%) had mutations in genes responsible for NS. All the identified mutations were previously reported. The descriptions of identified mutations are listed in Table 2. Most mutations were found in PTPN11 (35.4%), SOS1 (14.6%), and RAF1 (12.5%) (Table 2).
| Gene | No. | Frequency (%) |
|---|---|---|
| PTPN11 | 17 | 35.4 |
| SOS1 | 7 | 14.6 |
| RAF1 | 6 | 12.5 |
| KRAS | 5 | 10.4 |
| BRAF | 2 | 4.2 |
| RIT1 | 2 | 4.2 |
| negative | 9 | 18.8 |
| Total | 48 | 100 |
Mutation-positive rate: 81.3%
We evaluated clinical features in genetically diagnosed NS patients and found that cardiovascular abnormalities were the most common symptoms (87.2%), which included pulmonary stenosis (PS), hypertrophic cardiomyopathy (HCM), and atrial septal defect (ASD) (Tables 1 and 3). PS was prevalent in patients harboring mutations in PTPN11 (58.8%), KRAS (60%), or SOS1 (57.1%). HCM was found in all patients with mutations in RAF1, whereas only 2 patients with PTPN11 gene mutations (11.8%) exhibited HCM (Tables 1 and 3). The findings of electrocardiogram (ECG) and the age of cardiac operation are also described in Table 1. The second-most prevalent clinical feature was short stature, and this was found in 27 patients (69.2%). Short stature was more common in patients harboring mutations in PTPN11 (82.4%), RAF1 (83.3%), and KRAS (100%) than in those with SOS1 gene mutations (28.6%) (Tables 1 and 3). Mental retardation was found in 24 patients (66.7%) (Tables 1 and 3). Pectus deformities such as pectus excavatum, pectus excavatum, shield-shaped chest and barrel-shaped chest were noted in 8 of 24 subjects (33.3%). Cryptorchidism was observed in 8 of 19 males (42.1%) with 7 patients possessing mutations in PTPN11 (Tables 1 and 3). Neurological disorders including epilepsy and craniosynostosis were noted only in mutation-positive patients, but the prevalence was not significantly different from those without mutations (Tables 1 and 3). Craniosynostosis has been recently implicated as a comorbidity of NS [23], and 5 patients (3 with PTPN11, 1 with KRAS, and 1 with SOS1 mutations) were diagnosed as having craniosynostosis (Tables 1 and 3). Twelve of 35 patients (34.3%) exhibited otolaryngeal diseases including hearing disorders, otitis media, enlarged tonsil, and adenotrophy (Tables 1 and 3). Among them, hearing loss was most prevalent and its type and severity were described in Table 1.
| Mutation (+) | Mutation (–) | p-value | ||
|---|---|---|---|---|
| Cardiovasucular abnormalities | 34/39 (87.2%) | 7/9 (77.8%) | 0.44 | |
| PS | 22/39 (56.4%) | 5/9 (55.6%) | 0.96 | |
| ASD | 11/39 (28.2%) | 5/9 (55.6%) | 0.12 | |
| HCM | 14/39 (35.9%) | 2/9 (22.2%) | 0.43 | |
| Short stature | 27/39 (69.2%) | 6/9 (66.7%) | 0.88 | |
| Pectus deformity | 8/24 (33.3%) | 2/5 (40.0%) | 0.78 | |
| Mental retardation | 24/36 (66.7%) | 6/8 (75.0%) | 0.65 | |
| Neurological diseases | epilepsy | 6/37 (16.2%) | 0 (0%) | 0.20 |
| craniosynotosis* | 5/37 (13.5%) | 0 (0%) | 0.24 | |
| Otolaryngial disease | hearing loss | 8/35 (22.9%) | 1/9 (11.1%) | 0.35 |
| Urological disease** | cryptorchidism | 8/19 (42.1%) | 3/6 (50%) | 0.73 |
*: suspected cases are included
**: male patients are analyzed
To evaluate the clinical characteristics of NS patients with mutations, each clinical feature was compared between mutation-positive and mutation-negative patients by Chi-square test, and there were no significant differences between the groups (Table 3).
Clinical characteristics in NS patients with PTPN11 mutationsTo assess the clinical characteristics of NS patients with PTPN11 mutations, each clinical feature was compared between PTPN11 mutation-positive and mutation-negative patients by Chi-square test. Although the prevalence of PS and ASD was not different between the two groups, the prevalence of HCM was significantly lower in PTPN11 mutation-positive subjects (Table 4). The frequency of short stature and cryptorchidism was higher in PTPN11 mutation-positive subjects, but the difference was not significant (Table 4).
| PTPN11 mutation (+) | PTPN11 mutation (–) | p-value | ||
|---|---|---|---|---|
| Cardiovasucular abnormalities | 15/17 (88.2%) | 26/31 (83.9%) | 0.68 | |
| PS | 10/17 (58.8%) | 17/31 (54.8%) | 0.79 | |
| ASD | 7/17 (41.2%) | 9/31 (29.0%) | 0.39 | |
| HCM | 2/17 (11.8%) | 14/31 (45.2%) | 0.019 | |
| Short stature | 14/17 (82.4%) | 19/31 (61.3%) | 0.13 | |
| Pectus deformity | 6/12 (50.0%) | 4/17 (23.5%) | 0.14 | |
| Mental retardation | 12/16 (75.0%) | 18/28 (64.3%) | 0.46 | |
| Neurological diseases | epilepsy | 3/17 (17.6%) | 2/29 (6.9%) | 0.26 |
| craniosynotosis* | 3/17 (17.6%) | 2/29 (6.9%) | 0.26 | |
| Otolaryngeal disease | hearing loss | 5/17 (29.4%) | 5/27 (18.5%) | 0.40 |
| Urological disease** | cryptorchidism | 7/11 (63.6%) | 4/14 (28.6%) | 0.08 |
*: 1 suspected case is included
**: male patients are analyzed
We investigated whether there was any clinical characteristics in NS with p.N308 mutation in PTPN11 gene because this mutation was most prevalent in this study. For this purpose, clinical characteristics of NS with p.N308 mutation in PTPN11 gene were compared to those who possessed PTPN11 mutations other than p.N308. As shown in Table 5, clinical characteristics were not different between two groups except for the reduced prevalence of hearing loss in NS patients with p.N308 mutation.
| PTPN11 mutation | p-value | |||
|---|---|---|---|---|
| p.N308 | others | |||
| Cardiovasucular abnormalities | 8/8 (100%) | 7/9 (77.8%) | 0.16 | |
| PS | 5/8 (62.5%) | 5/9 (55.6%) | 0.77 | |
| ASD | 4/8 (50.0%) | 5/9 (33.3%) | 0.49 | |
| HCM | 2/8 (25.0%) | 0/9 (0%) | 0.11 | |
| Short stature | 7/8 (87.5%) | 7/9 (77.8%) | 0.60 | |
| Pectus deformity | 3/6 (50.0%) | 3/6 (50.0%) | 1.00 | |
| Mental retardation | 6/8 (75.0%) | 6/8 (75.0%) | 1.00 | |
| Neurological diseases | epilepsy | 2/8 (25.0%) | 1/9 (22.2%) | 0.45 |
| craniosynotosis* | 1/8 (12.5%) | 1/9 (11.1%) | 0.93 | |
| Otolaryngial disease | hearing loss | 0/8 (0%) | 5/9 (55.6%) | 0.012 |
| Urological disease** | cryptorchidism | 2/5 (40.0%) | 5/6 (88.3%) | 0.14 |
*: suspected cases are included
**: male patients are analyzed
We evaluated the present height SDS in genetically diagnosed NS patients and analyzed its relationship to genotype. The mean present height SDS in each genotype is shown in Fig. 1. Statistical analysis using one-way ANOVA was performed to compare the height SDS among genotypes containing more than 3 patients, which revealed that patients showed significantly higher and lower height SDS with mutations in SOS1 and KRAS, respectively. Target height was available in 16 subjects as shown in Table 1.
Present height SDS in each genotype
Present height SDS in each genotype is shown. Statistical analysis was performed using ANOVA.
Most patients were born full term, and the gestational week at delivery was not different among genotypes. Birth weight SDS and length SDS was in the normal range in most patients. Statistical analysis using one-way ANOVA was performed to compare birth weight SDS and length SDS among genotypes containing more than 3 patients, which revealed that neither birth weight SDS nor length SDS was different among genotypes (Fig. 2). Pearson’s correlation analysis in genetically diagnosed patients revealed no association between height SDS at birth and present height SDS (r = 0.30, p = 0.08) (Fig. 3A).
Birth measurements in each genotype
Weight SDS (A) and height SDS (B) at birth are shown. Statistical analysis was performed using ANOVA.
Present height SDS is positively associated with height SDS at 12 months old
Correlation between present height SDS and birth height SDS (A) or height SDS at 12 months (B) was evaluated by Pearson’s correlation analysis. All the clinically diagnosed NS patients were included for analysis.
Linear growth during the first 12 months was evaluated in 19 patients including 8 with PTPN11, 2 with RAF1, 5 with SOS1, 1 with KRAS, 2 with BRAF, and 1 with RIT1 mutations (Table 6). Average height SDS at 12 months old was shown in Fig. 4A. Statistical analysis using a Student’s t-test was performed to compare the values between genotypes containing more than 3 patients and revealed that patients with SOS1 mutations showed smaller decreases in height SDS than those with PTPN11 mutations during this period (Fig. 4A and B). Pearson’s correlation analysis in genetically diagnosed patients revealed a positive association between present height SDS and height SDS at 12 months old (r = 0.88, p < 0.0001) (Fig. 3B), suggesting that growth during the first year of life is important in determining height later in life.
| No. | Gene mutation | Sex | Ht SDS at birth | Ht SDS at 12 months | ΔHt SDS during 12 months |
|---|---|---|---|---|---|
| 2 | PTPN11 | F | 0.5 | –2.6 | –3.1 |
| 3 | PTPN11 | M | 1.0 | –1.7 | –2.7 |
| 4 | PTPN11 | F | –0.8 | –2.0 | –1.2 |
| 6 | PTPN11 | M | –1.5 | –2.4 | –0.9 |
| 7 | PTPN11 | M | –0.1 | –3.1 | –3.0 |
| 8 | PTPN11 | M | 1.7 | –2.6 | –4.3 |
| 9 | PTPN11 | M | –1.8 | –5.7 | –3.9 |
| 10 | PTPN11 | F | –0.1 | –2.0 | –1.9 |
| 18 | RAF1 | F | –0.3 | –3.0 | –2.7 |
| 19 | RAF1 | F | –0.5 | –2.8 | –2.3 |
| 24 | SOS1 | M | 0.3 | 0.2 | –0.1 |
| 25 | SOS1 | F | 0.6 | –0.7 | –1.3 |
| 26 | SOS1 | F | –1.0 | –1.6 | –0.6 |
| 27 | SOS1 | M | 0.1 | –1.7 | –1.8 |
| 28 | SOS1 | M | 0.8 | –1.4 | –2.2 |
| 31 | KRAS | F | 0.2 | –2.8 | –3.0 |
| 36 | BRAF | F | 0.8 | –2.0 | –2.8 |
| 37 | BRAF | F | 0.2 | –2.4 | –2.6 |
| 38 | RIT1 | F | –0.2 | 0.4 | 0.6 |
Infantile growth is impaired in NS
Height SDS at 12 months (A) and infantile growth during the first 12 months in each genotype were determined.
Twelve patients reached an adult height. Height SDS was in the normal range in 3 patients (2 with PTPN11 and 1 with RIT1 mutations) (Table 7). The remaining 9 patients had a short stature with a height SDS ranging from –2.0 to –4.8 SD. Two patients with KRAS mutations exhibited a severe short stature with a height SDS below –4.0 SD. Target height was available in 3 subjects (Table 7).
| No. | Genotype | Sex | Adult height (cm) | Adult height SDS | Target height SDS |
|---|---|---|---|---|---|
| 12 | PTPN11 | F | 158.0 | 0.1 | NA |
| 14 | PTPN11 | M | 149.8 | –3.6 | NA |
| 16 | PTPN11 | M | 161.1 | –1.5 | –0.7 |
| 17 | PTPN11 | F | 148.1 | –2.0 | –0.9 |
| 22 | RAF1 | M | 158.4 | –2.1 | NA |
| 23 | RAF1 | M | 155.5 | –2.7 | NA |
| 30 | SOS1 | M | 159.8 | –2.2 | NA |
| 33 | KRAS | F | 147.3 | –2.5 | NA |
| 34 | KRAS | M | 145.5 | –4.4 | NA |
| 35 | KRAS | F | 133.7 | –4.8 | NA |
| 39 | RIT1 | F | 161.2 | 0.7 | –0.4 |
| 48 | Negative | M | 160.1 | –2.2 | NA |
Adult height is defined as height velocity being 2 cm/yr or less.
NA, not available
After GH treatment was available for NS patients with short stature as of 2017 in Japan, total of 7 subjects received GH treatment for at least 1 year. None had GH deficiency based on GH provocation test. Although the staring dose of GH for NS with short stature is 0.23 mg/kg/week based on the recommendation from Japanese Society for Pediatric Endocrinology, 3 subjects received GH at a dose of 0.46 mg/kg/week because GH treatment in these subjects was initiated as part of clinical trial for GH treatment for NS with short stature. All of them showed responses to GH treatment with various extent (Table 8). IGF-1 levels were also increased in response to GH treatment in all the subjects (Table 8).
| Case No. | Sex | Gene | Starting age | GH dose (mg/kg/week) | pre Ht SDS | 1 yr Ht SDS | ΔHt SDS | pre IGF-1 SDS | 1 yr IGF-1 SDS | ΔIGF-1 SDS |
|---|---|---|---|---|---|---|---|---|---|---|
| 3 | M | PTPN11 | 4y | 0.46 | –2.2 | –1.2 | 1.0 | –1.8 | –1.2 | 0.6 |
| 5 | M | PTPN11 | 3y | 0.46 | –3.0 | –1.4 | 1.6 | –1.2 | 1.6 | 2.8 |
| 6 | M | PTPN11 | 5y | 0.23 | –2.1 | –1.4 | 0.7 | –0.2 | 1.8 | 2.0 |
| 7 | M | PTPN11 | 6y | 0.46 | –2.5 | –1.5 | 1.0 | –2.4 | –0.4 | 2.0 |
| 8 | M | PTPN11 | 9y | 0.23 | –2.7 | –2.3 | 0.4 | –2.6 | –1.8 | 0.8 |
| 10 | F | PTPN11 | 7y | 0.23 | –2.2 | –1.9 | 0.3 | –1.6 | 0.0 | 1.6 |
| 18 | F | RAF1 | 3y | 0.23 | –3.5 | –2.2 | 1.3 | –2.5 | –1.0 | 1.5 |
We examined genotype-phenotype correlations in Japanese NS patients, and found that the mutation-positive rate was 81.3%; mutations in PTPN11 were the most prevalent, and we found a greater prevalence of KRAS mutations than that of previous studies [2, 9]. The difference in the prevalence of KRAS mutations may be caused by a small sample size because a previous study that analyzed 65 clinically diagnosed Japanese NS patients revealed only 1 patient with KRAS mutations [24]. Because all the genes analyzed were components of the RAS/MAPK signaling pathway, we also evaluated whether patients with mutations in the RAS/MAPK signaling pathway possessed any clinical characteristics, but did not detect any differences between mutation-positive and mutation-negative subjects. These findings suggest that mutation-negative NS patients have mutations in molecules involved in or modulating the activity of the RAS/MAPK pathway. In support of this notion, recently identified genes, such as PPP1CB [25] and LZTR1 [26], have been involved in this pathway [27].
Short stature is one of the main findings of NS. The growth characteristics of NS include normal values of birth weight and height with catch-down growth during infancy [28]. The growth rate during childhood is relatively maintained, and the onset of the pubertal spurt is slightly delayed, which improves the final height in some patients [28]. The height SDS at 1 year of age was significantly correlated with the present height SDS, which suggests that growth during infancy is a major determinant for adult height in NS. Although the mechanisms of infantile growth retardation are not well understood, an increased prevalence of feeding difficulties partly because of gastroesophageal reflux disease may result in malnutrition and cause growth retardation during this period. Therefore, strategies that help NS patients specifically during infancy may increase adult height. In addition to nutritional aspects, genotype may also affect growth in NS patients. Present height SDS was significantly affected by genotype such that patients with SOS1 and KRAS gene mutations showed higher and lower height SDS, respectively [19, 29]. Although the mechanisms of genotype-specific regulation of growth in NS is not fully understood, the fact that patients with SOS1 mutations displayed better growth during the first year of life may again highlight the importance of growth during infancy to achieve better adult height. It is still unclear why patients with SOS1 mutations showed better postnatal growth; however, since the severity of mental retardation has been implicated to be mild in NS patients with SOS1 mutations [2, 30], this may be partly responsible for better infantile growth in these patients. Indeed, the severity of mental retardation was mild in patients with SOS1 mutations in the current study (data not shown), although the frequency of mental retardation was high (5 of 6 patients with SOS1 mutations).
Because PTPN11 mutations were the most prevalent in our study, we compared clinical characteristics between PTPN11 mutation-positive and mutation-negative subjects. With respect to cardiac anomalies, the prevalence of HCM was significantly low in PTPN11 mutation-positive patients, whereas the prevalence of PS and ASD was not different, which is in part similar to previous papers [16, 18, 31-33]. The prevalence of short stature was increased in PTPN11 mutation-positive patients in the current study, but the difference did not reach statistical significance. Because patients with PTPN11 mutations have lower IGF levels and exhibit impaired growth in response to GH therapy, GH resistance may be a characteristic feature in NS patients with PTPN11 mutations and contribute to the development of short stature in these patients [16, 18]. The prevalence of cryptorchidism showed greater in patients with PTPN11 mutations although the difference did not reach statistical significance. Because Japanese population-based analysis also revealed a trend toward increased prevalence of cryptorchidism in patients with PTPN11 mutations [31], this may be a characteristic feature in Japanese NS patients with PTPN11 mutations; however, further analysis is needed.
Since p.N308 mutation in PTPN11 gene was most prevalent, we analyzed mutation site-specific genotype-phenotype correlation in PTPN11 gene, and found that hearing loss was not noted in those with p.N308 mutation; however, based on the previous finding that hearing loss was observed in NS patients with PTPN11 p.N308 mutation [34], p.N308 mutation in PTPN11 gene is not specific to hearing impairment and the current findings may be biased by the small sample size. Animal study revealed that accumulation of macrophage was observed in the organ of corti of a LEOPARD syndrome mouse model [35], suggesting that activation of RAS/MAPK pathway is responsible for hearing impairment. In addition, the prevalence of mental retardation was not decreased in those with p.N308 mutation although previous paper showed that those with p.N308D showed better mental development [18]. These findings clearly indicate that further analysis with larger scale is required to unravel mutation site-based genotype-phenotype association.
This study has several limitations. First, mutational analysis did not include all the responsible genes such as PPP1CB [25] and LZTR1 [26], and therefore the estimation of the mutation-positive rate may not be accurate. Second, we did not evaluate adult height in these subjects, and the effect of each mutation on adult height is not fully understood. In addition, target height-based analysis was not performed because target height was not available in most of the subjects. Third, because of the small number of subjects, we did not analyze the sex-specific effects of each mutation. Fourth, due to the large variation in age at analysis (from 1 to 39 years old), the prevalence of complications may be underestimated. To overcome these limitations, additional analyses are clearly required.
In conclusion, we analyzed the genotype-phenotype correlation in Japanese NS patients and found that the mutation-positive rate was similar to previously reported papers. We also found that impaired growth during infancy was strongly correlated with present height SDS. Although further analyses including responsible gene-based genotype-phenotype correlation analysis with functional assessments are needed to clarify genotype-phenotype correlations in Japanese patients, these findings may enable the use of genetic information to predict the clinical course and may allow for genotype-based medical interventions.
YS and MK conceived the project and designed the research. All the authors performed data collection and analyses. TN and YA performed genetic analysis. YS and MK wrote the manuscript.
The authors have nothing to disclose.
