Noonan syndrome (NS; OMIM#163950) is an autosomal dominant disorder characterized by craniofacial dysmorphism, such as short webbed neck (68.4%), downslanted palpebral fissures (46.6%), low posteriorly rotated ears (43.3%), and microcephaly (32.5%). Other features include congenital heart diseases (CHD) (83.8%), thoracic and musculoskeletal deformities (short stature) (77.4%), intellectual disabilities (ID) (20%) [1,2,3], and other additional extracardiac features. The incidence of NS is estimated to be 1 in 1000-2500 live births [4,5].

NS is a heterogeneous condition, with variable phenotypic expression and severity. The syndrome is associated with pathogenic variants in multiple genes in the Ras/Mitogen-Activated Protein Kinase (RAS-MAPK) pathway, which regulates growth and differentiation, leading to dysregulation. More than eight different genes associated with NS, such as PTPN11 (61%), SOS1 (12%), RAF1 (7%), RIT1 (5%), LZTR1 (4%), and other implicated genes, including SOS2, BRAF, KRAS, MAP2K1, MRAS, NRAS, RASA2, and RRAS2, are required to diagnose NS.

Pathogenic variants in the PTPN11 gene are the major cause of NS, with missense mutations responsible for about 50% of NS cases. This gene encodes the tyrosine protein phosphatase non-receptor (SHP2), which is involved in Extracellular Signal-Regulated Kinase (ERK) activation via the RAS-MAPK pathway [6]. PTPN11 is located on chromosome 12q24 and contains 15 exons, encoding a total of 593 amino acids. Variants in PTPN11 are most frequently found in exons 3 and 8 [7]. The hotspot variant in the PTPN11 gene associated with NS is c.922A>G (p.Asn308Asp) in exon 8. This variant has been widely studied and reported across a range of populations with prevalence of 16.3% to 43%. The second, c.1510A>G (p.Met504Val) in exon 3, is less prevalent, with a range of 4.2% to 16.3% [1]. Other recurrent variants include c.417G>C (p.Glu139Asp) in exon 8, c.836A>G (p.Tyr279Cys) in exon 7, and c.1403C>T (p.Thr468Met) in exon 13, which are also often found in populations [8].

The PTPN11 gene is expressed in various tissues and plays a crucial role in regulating eukaryotic cell responses to multiple extracellular signals, such as hormones, cytokines, and growth factors. Mutations in PTPN11 can be inherited in an autosomal dominant manner or occur de novo. The mutations involved in NS are considered gain-of-function mutations that cause inappropriate prolongation of the RAS/MAPK signaling pathway. The prolonged RAS/MAPK pathway leads to the pleomorphic characteristics in NS [9].

NS is characterized by a broad spectrum of symptoms and physical features that vary greatly in range and severity, and can change with age. The most consistent features are wide-set eyes, low-set ears, short stature, and pulmonary valve stenosis [9,10]. The diagnosis of NS syndrome depends on the identification of characteristic clinical features [10,11].

Clinical diagnosis of NS based on the diagnostic criteria for NS. Until recently, diagnosis was made based on clinical features. Molecular genetic testing can confirm approximately 70% of cases [10]. There are major and minor criteria, along with some features, for establishing the diagnosis of NS (Table 1). The features are facial features, cardiac features, height, chest wall shape, family history, and other characteristics such as ID, cryptorchidism, and lymphatic vessel dysplasia [11].

We included 29 patients who visited the Cytogenetic and Molecular Laboratory, Biomedical Research Center Laboratory (CEBIOR), Faculty of Medicine Universitas Diponegoro, Semarang, Indonesia. The inclusion criteria for subjects are age 0-18 years old who were diagnosed with NS based on Van der Burgt criteria (Table 1) and were clinically confirmed by an experienced pediatrician.

To the best of our knowledge, this is the first study involving a relatively large NS patient cohort in Indonesia. Missense mutations in PTPN11 cause NS and involve a gain-of-function mechanism. In the present study, we identified PTPN11 changes in 18 (62%) out of 29 patients, all of whom had sporadic NS. These findings are limited as we performed only on selected exons, including PTPN11 exons 3, 4, 8, 11, and 13. The result is consistent with previous studies, which reported that PTPN11 pathogenic variants were present in 50% of NS cases [24,25,26,27]. Other genes responsible for NS, such as SOS1, SOS2, BRAF, KRAS, MAP2K1, MRAS, NRAS, RAF1, RASA2, RIT1, RRAS2, and LZTR1, may still be found in other patients. Further examination to study these genes and exons will be planned in the future as well.

Six of the 18 pathogenic variants identified in this study were previously reported missense changes. The majority of variants were found in exon 8, as reported in earlier studies, accounting for 72.2% of the PTPN11 pathogenic variants (Table 3). In this study, 18 (62%) of those with PTPN11 pathogenic variants had variants in exons 3, 8, and 13; this proportion is consistent with that observed in most studies [13,28,29,30,31].

However, one pathogenic variant (c.140G>A p.(Arg47Lys)) represents a novel finding within the N-terminal SH2 domain (N-SH2). N-SH2 plays a critical regulatory role as a key regulatory domain of the protein. This variant has not been previously reported in major population databases and literature [6]. Variants affecting the N-SH2 domain of PTPN11 are associated with NS due to a gain-of-function that destabilizes the autoinhibitory interaction between the N-SH2 and Protein Tyrosine Phosphatase (PTP) domains. Thus, SHP2 becomes excessively active, leading to hyperactivation of the RAS-MAPK pathway and dysregulated developmental signaling [32]. In silico predictive tools suggest that the variant is probably damaging, and conservation analysis demonstrates it is highly conserved across species, indicating potential functional importance [33].

The missense variant in c.907G>A p.(Asp303Asn) was the most common pathogenic variant we identified. This variant was previously reported in a case of Kawasaki disease in NS by Takai et al. In silico analysis predicted that it was “probably damaging” by Poplyphen2 with a prediction score of 0.533, adding further evidence of the occurrence of this variant in another NS patient [15]. A missense variant, c.922A>G (p.Asn308Asp), found in one patient, is consistent with earlier studies on PTPN11 pathogenic variants, which identified c.922A>G as a mutation hotspot for NS-causing missense changes [8,13,14].

Sequence variant c.907G>A p.(Asp303Asn) is likely to represent a non-synonymous single-nucleotide polymorphism (nsSNP) that causes NS in Javanese, Southeast Asian, and Indonesian populations. In our case, we have collected data on family trees; however, we have not found evidence of familial segregation. Therefore, functional studies are needed for this variant. The pathogenic nsSNPs of PTPN11 c.907G>A p.(Asp303Asn) were analyzed by five in-silico pathogenicity tools, such as PolyPhen, REVEL, SIFT, FATHMM Pred, and MutationTaster. The results support a deleterious effect on protein function in some predictors, but with the REVEL score of 0.533, it is not convincing enough to categorize it as likely pathogenic. With only one previous case report of a Noonan Syndrome patient with this variant [15], we classified it as a variant of unknown significance (VUS), which will need further evidence to reclassify it, according to the American College of Medical Genetics and Genomics (ACMG) guidelines [34]. Considering that about 30% of nsSNPs may influence clinical phenotypes by altering post-translational modifications (PTMs), protein stability, and protein-protein interactions, functional studies and clinical correlation are recommended for definitive pathogenicity confirmation [35,36].

The SNPs in the coding region of human genes were associated with genetic disorders. A large number of SNPs have been reported in the database, making it difficult to screen them all for a particular phenotype. Computational analysis tools help narrow down and examine pathogenic SNPs for specific genetic disorders, thereby minimizing risk [37,38].

In many previous studies, genotype-phenotype correlations have been widely investigated. However, the result obtained is that within the same pathogenic variant location, a wide range of clinical variability is observed [24]. Several studies describe the role of SHP2 in regulating various developmental processes and the possible causes of this variation [39,40,41].

Variability in craniofacial features may vary due to subjective assessment. Low-set posteriorly rotated ears (83.3%) are the most commonly observed in our study, followed by microcephaly, downslanted palpebral fissure, and short-webbed neck (50%). Hypertelorism and depressed nasal bridge are present in eight (44.4%) patients, while prominent eyes are present in seven (38.9%) of the 18 cases. The mean age is 1 year 11 months, with children being the majority of cases, consistent with the literature on craniofacial features in childhood with NS. In the childhood category, the head is relatively abnormal in shape, with low-set, posteriorly rotated ears, downslanted palpebral fissures, a wide forehead, epicanthal folds, a short-webbed neck, and sternal deformities (pectus carinatum or excavatum). Hypertelorism, ptosis, or thick, hooded eyelids are characteristics. The nose is short and wide with a depressed root [42]. Thereafter, due to age changes, the craniofacial features become less noticeable in adulthood.

Severe short stature (HAZ below -3 SD) and short stature (HAZ below -2 SD or at the 3rd percentile) were seen in 13 (72.2%) of 18 patients with PTPN11 pathogenic variants. Proportionate short stature is present in more than 50% of cases. The molecular mechanism by which NS-causing SHP2 mutants induce growth retardation had been reported previously [43]. SHP2 mutations in NS by a postreceptor signaling defect cause mild Growth Hormone (GH) resistance, which is partially compensated by increased GH secretion. In children with SHP2 mutations who have a poor response to rhGH, this defect may contribute to the short stature phenotype [44]. Growth delay was associated with low Insulin-like Growth Factor 1 (IGF-1) levels during the early postweaning growth phase. Impaired IGF-1 production contributes to growth retardation in NS. This is in agreement with clinical data showing that NS patients, notably those carrying a PTPN11 mutation, display low IGF-1 levels [39]. GH treatment in NS is thought to be beneficial because the dysmorphic findings are similar to those in Turner syndrome. Therefore, GH has been administered at the same dose used in the treatment of Turner syndrome [45,46].

NS is the second most common syndromic cause of congenital heart disease, exceeded in prevalence only by trisomy 21 [23]. Clinical manifestations of CHD problems are the most common reasons that prompt patients to see a doctor. In this study, CHD was found in 15 patients (83.3%) with PTPN11 pathogenic variants. Pulmonary valve stenosis, seen in 9 patients (50%), was the most common finding in the present study, followed by atrial septal defects in 6 patients (33.3%), either isolated or combined with other types. Compared to the data from the previous study, NS patients with the PTPN11 pathogenic variant are more likely to be associated with pulmonary valve stenosis or ASD [47]. PTPN11 encodes the Src homology-2 domain-containing protein tyrosine phosphatase and is reported to be associated with enlargement of the aortic annulus and aortic root [48,49].

Thoracic and musculoskeletal deformities of NS consist of insufficient and/or delayed growth, causing short stature, and axial skeletal deformities, including sternal deformities, vertebral anomalies, cubitus valgus, and finger-toes abnormalities. Sternal deformities, such as pectus carinatum and/or excavatum, were presented in 10 (55.6%) of the 18 cases with PTPN11 pathogenic variants. Four patients (27.8%) had widely-spaced nipples, and the chest circumference was measured across the internipple line. The internipple index was calculated according to the formula: internipple distance (cm) multiplied by 100 and divided by chest circumference (cm). The internipple distance and chest circumference increased with age. The internipple index in the neonatal period was the highest among all ages (26.4 ± 1.6 for males and 26.3 ± 2 for females), and will gradually decrease until the age of four years (23.8 ± 1.2 for males and 23.8 ± 1.4 for females), thereafter it would be relatively constant through the age of 18 years in males and the age of 11 years in females [48].

Neurological manifestations and cognitive and behavioral changes in individuals with NS are highly variable. Heterogeneity in cognitive abilities within RAS-MAPK signaling pathway syndromes has been observed, which depends on the affected gene and the variant type. IQ tests were not examined; instead, the cognitive & behavioural changes were assessed by a Social Pediatric Consultant using the Denver II as a screening tool to identify potential ID. This assessment evaluated four developmental areas: personal-social, fine-motor-adaptive, language, and gross-motor skills. Eight patients (44.4%) were found to have global developmental delay, as their screening test results did not align with their actual age. In these patients, neurodevelopmental and hearing function evaluations should be performed [50].

Cryptorchidism may be present in males with NS and have an increased risk of infertility. While females with NS generally have normal puberty and fertility, males may experience delayed puberty and reduced fertility due to cryptorchidism and other potential gonadal issues. Male gonadal dysfunction has also been reported and is suggested to be caused by primary Sertoli cell dysfunction rather than cryptorchidism [51]. Among the nine male patients in this study, four have cryptorchidism, although all the patients have undergone orchidopexy as corrective surgery.

Lymphatic vessel dysplasia with a clinical lymphatic abnormality is a major feature of NS. Six NS patients (33.3%) with PTPN11 pathogenic variants had lower limb lymphedema. Lower limb lymphedema and genital lymphedema are the two most reported types of lymphedema that develop after birth [52]. The presentation of lymphedema in NS may vary, including differences in timing and resolution of edema at different ages. Haque et al. [53] reported prenatal and postnatal onset of lymphatic abnormalities in NS, whereas postnatal onset might result in a milder phenotype.

A broad spectrum of ophthalmologic features is found in NS, with downslanted palpebral fissures (50%) being the most common finding in PTPN11 pathogenic variants, followed by depressed nasal bridge, hypertelorism (44.4%), and prominent eyes (38.9%). Marin et al. [54] reported that the most common ophthalmologic feature finding (74%) in patients with PTPN11 pathogenic variants was downslanted palpebral fissures. Refractive errors are not examined. Based on the literature, refractive errors and ptosis are often detected and reported in patients with PTPN11 pathogenic variants [55].

Several hematological disorders are predisposed to occur in NS, including transient monocytosis, thrombocytopenia, and myeloproliferative disorder [56]. The most common haematologic disorders are coagulation defects, manifested as bleeding abnormalities [57]. In this study, one NS patient with a PTPN11 pathogenic variant had severe anemia, while other patients had haemangioma and juvenile myelomonocytic leukemia. Several hematological cancers have been reported in patients with NS during childhood, including juvenile myelomonocytic leukemia, acute myelogenous leukemia, and B-cell acute lymphoblastic leukemia [57].

Despite a few studies reporting an autoimmune thyroiditis in NS, an earlier study showed an association between hypothyroidism and NS. In a patient with NS, primary hypothyroidism is an uncommon condition that may develop either separately or in conjunction with NS. Although the precise etiology of this condition is unknown, age, excessive iodine intake, and heredity can make an individual more susceptible. One NS patient in this study was found to have hypothyroidism and undertreatment with Euthyrax. A multidisciplinary approach is required for these individuals with comorbidities or related conditions to NS [58].

Gastrointestinal manifestations such as liver problems, including elevated liver enzymes, autoimmune hepatitis, and even human hepatocellular carcinoma (HCC), can occur in NS due to its genetic basis affecting the RAS-MAPK pathway, which is commonly seen in other RASopathies like progressive familial intrahepatic cholestasis (PFIC) [59]. We reported one NS patient with a pathogenic PTPN11 variant with cholestasis. Liver problems in NS are less frequent; a previous study by Rippert [60] revealed a strong association between NS and neonatal liver disease, particularly neonatal hyperbilirubinemia and cholestatic disease. Since the pathogenesis of these conditions has not been definitively linked to NS, understanding the spectrum of liver phenotypes in NS remains a subject of ongoing investigation.

NS is linked to various orodental manifestations reported in several studies. High-arched palate, micrognathia, and macrodontia, which can result in malocclusion and misalignment of teeth, have been reported repeatedly in case reports and retrospective studies [61]. High arched palate and caries dentis are common dental problems in NS.

Cutis marmorata teleangiectatica congenita (CMTC), a cutaneous or ectodermal manifestation of NS, was found in one patient in this study. CMTC is a possible associated anomaly in NS, even though it is not a primary feature. Cutaneous findings are common in NS with BRAF, KRAS, and SHOC2 variants [62].

The study’s retrospective design had some limitations. We tried to combine the correlations between genotypes and phenotypes based on follow-up results in the medical records from the last examination. Due to the high cost and the fact that genetic testing is not covered by national health insurance, genetic testing has only been performed on selected exons, those frequently reported to cause NS mutations, which were evaluated. Moreover, the relatively small cohort may not adequately represent the broader population variability and may have biased the results. However, this study is, to the best of our knowledge, the first to explore genotype-phenotype correlations of NS. in the Indonesian population, which might be a basis for further study in NS.

NS has broad clinical manifestations, and each patient presents with mixed clinical features. Clinicians need to raise awareness of suspecting NS, since the incidence rate is relatively high, and be concerned about comorbidities. Early diagnosis can be achieved by maintaining a high index of suspicion in children with short stature, ID, CHD, and musculoskeletal deformities that have a distinctive facial dysmorphism at any age. Whole exome sequencing or gene panel is the gold standard for NS and other RASopathies. However, in this developing country, Indonesia, where most of the cases come from families with low economic backgrounds, genetic testing is very expensive and not covered by government health insurance. In these situations, based on the results of our study, we might suggest molecular testing, such as Sanger sequencing, for patients clinically suspected of having NS, performed only at several mutation hotspots, such as exons 3, 8, and 13.