In this medical record review the clinical data of patients presenting with Noonan syndrome and related disorders in our genetic clinic from 2014 through 2019 was collected on a structured defined proforma. We excluded patients with neurofibromatosis as they form a distinct, easily identifiable, group. Informed consent was taken at the time of evaluation and molecular testing from all patients/parents included in the study. The PTPN11 gene was sequenced or next-generation sequencing (NGS) using a panel/clinical-exome approach on Illumina HiSeq2500 was performed. All the molecular variants were classified according to the recommended method of the American College of Medical Genetics and Genomics [2]. Patients who did not undergo molecular testing were classified according to the predominant phenotype. The clinical data is represented as proportions for frequency of phenotypic features and mutations.

The study cohort included 30 patients, (23 males); 22 of which (16 males) were diagnosed with Noonan syndrome, five patients with CFC and three patients with NSLAH. The mean age of patients in the cohort was 7 years [range 4 months to 23 years]. Mutations were identified in 27 patients. In two patients only PTPN11 sequencing was done, which was negative and one patient did not consent for molecular testing.

The age of diagnosis of Noonan syndrome patients ranged from 4 months to 23 years. The predominant clinical features were cardiac disease (82%), short stature (77%), facial dysmorphism (64%), skeletal features like scoliosis, webbed neck, chest defects (pectus and wide space nipples) (45%), mild developmental delay (27%), coagulation abnormalities (23%) and cryptorchidism (14%) (Table I). The commonest cardiac defect was pulmonary stenosis (39%, 7/18) followed by hypertrophic cardio-myopathy (33%, 6/18). Skin features like café au lait macules (size varying from 5-10 mm, more than three) and hyperkeratosis were present in 27% patients. One child presented at 3 months of age with juvenile myelo-monocytic leukemia (JMML) syndrome (Table I). Antenatal features of cystic hygroma, bilateral choroid plexus cyst and dilated single lymphatic sac were documented in one child with Noonan syndrome related short stature.

Facial dysmorphism was present in 14 (64%) patients. (Fig.1 a, b). The four most characteristic features (hyper-telorism, down-slanting palpebral fissures, ptosis, and low-set, posteriorly rotated ears) were present together in only four patients; 10 had atypical facies with one or two of the above dysmorphic features. Down-slanting palpebral fissures were seen in 64% and hypertelorism in 57%. Two patients had coarse facies and ectodermal features and were initially suspected as CFC syndrome but were later diagnosed as Noonan syndrome based on genetic testing (RAF1 and SOS2, respectively) (Fig. 1b).

Fig. 1 Variable facial dysmorphism in Noonan syndrome: (a) Boy with Noonan Syndrome with hypertelorism, ptosis, downslant palpebral fissures, low set posteriorly rotated ears (PTPN11, exon 3, c.218C>T), (b) Boy with Noonan syndrome with cardio-facio-cutaneous syndrome like phenotype – coarse face, woolly hair, ptosis, hypertelorism, low set posteriorly rotated ears, (SOS2, Exon 6, c.800T>G), (c) Boy with cardio-facio-cutaneous syndrome - coarse face, hypertelorism, downslant eyes, low set ears, coarse hair (BRAF, exon 15, c.1802A>T) and (d), (e) Boy with Noonan syndrome like disorder with loose anagen hair - coarse face, hypertelorism, downslant eyes, relative macrocepahly and the distinct sparse slow growing hair.

Five patients of CFC syndrome were identified. All had developmental delay, coarse facies and ectodermal findings (Web Table I) (Fig. 1c). All the three patients with NSLAH had mild developmental delay, coarse facies and sparse, slow growing hair (Fig. 1 d, e). One patient addi-tionally had a history of thrombotic stroke (Web Table I).

Of the 22 Noonan syndrome patients, mutations were present in 19 (86%) patients. These were present in PTPN11 (11/19), SOS1 (2/19), SOS2 (2/19), RIT 1 (2/19), KRAS (1/19) and RAF1 (1/19) genes. The mutations and related information are listed in Web Table II. All the identified mutations are previously reported. The two CFC syndrome patients had the most common BRAF mutation, c.770A>G, p.Gln257Arg. All three NSLAH patients harbored the recurrent SHOC2, c.4A>G, p.Ser2Gly mutation (Web Table I).

The clinical diagnosis of Noonan syndrome is traditionally on a gestalt recognition of the characteristic facial dysmorphism, cardiac malformations and short stature. Associated ectodermal features suggest CFC and NSLAH as the probable diagnosis [3,4]. In this cohort, Noonan syndrome was the commonest RASopathy (73%), followed by CFC (17%) and NSLAH (10%). The most consistent and typical facial features in the Noonan syndrome cohort were down-slanting palpebral fissures, ptosis and hypertelorism, similar to previous reports [5]. However, we also observed PTPN11 mutation positive Noonan syndrome with atypical facies, including only hypertelorism, down-slanting palpebral fissures or ptosis. Another set of patients with mutations in uncommon Noonan syndrome genes like RIT1, SOS1 and SOS2 had the typical NS facial phenotype. A CFC like phenotype was seen with mutations in RAF1 and SOS2 associated NS suggesting a phenotypic overlap between NS and CFC. As the facial profile in NS evolves with age, it alone may be insufficient to predict the genotype, but along with other systemic features, it can aid in the clinical diagnosis [6].

The predominant cardiac lesions in NS are pulmonary stenosis (PS) and hypertrophic cardiomyopathy (HCM). Early suspicion and echocardiography is important for appropriate management as PS and HCM in PTPN11 related NS are seldom rapidly progressive and fatal [7]. Short stature was another predominant phenotype observed in this study, which may be due to growth hormone (GH) deficiency, neurosecretory dysfunction, or GH resistance. GH therapy is approved for Noonan syndrome and should be initiated early [8].

Renal abnormalities are described in 10-11% of cases of Noonan syndrome [9]. In the present study one patient with KRAS associated NS (NS-3) had bilateral grade 5 vesicoureteric reflux (VUR) with hydronephrosis. VUR leading to hydronephrosis is previously unreported in Noonan syndrome. It reiterates the need for multi-organ screening in malformation syndromes for early detection and management, and prevention of related morbidity [10]. In one patient (NS-9, SOS2 mutation) with abnormal gait and brisk deep tendon reflexes, MRI brain showed bilateral thalamic hyperintensities. MRI changes in RASopathies are previously reported, but MRI brain is recommended only if there is abnormality neurological examination [12]. Bleeding abnormalities are reported in almost 43% patients of NS while on laboratory testing abnormal coagulation profile is described in upto 90% patients [13]. One patient with NSLAH had a history of thrombotic stroke. This previously unreported association is either incidental or a disease association and needs to be addressed in additional patient cohorts. Specific PTPN11 gene mutations predispose to an increased risk of JMML in NS patients [14], but they have a favorable prognosis and better outcomes, highlighting the importance of this correlation in management protocols [15].

A previous Indian study reported exons 3 and 13 of PTPN11 gene as the mutation hot spot in 11 Noonan syndrome patients [16]. Another study identified exons 3, 8 and 13 of PTPN11 gene with the maximum pathogenic variants in 107 Indian patients [17]. Exons 3, 8, 12 and 13 were the hotspots exons and the commonest mutation was a previously reported, c.218C>T in exon 3 in this series. Additionally, the recurrent SOS2, c.800T>A mutation of NS-9 was also present in two patients [18]. We observed that most mutations in Indian patients were similar to those reported in worldwide literature.

Limitations of this study include a small number of predominantly NS patients with less representation of CFC and NSLAH. Also the absence of longitudinal follow up data limits information on management outcomes and prognosis of the patients.

Noonan syndrome should be suspected in patients with short stature (cardiac malformations, primarily pulmonary stenosis and hypertrophic cardiomyopathy), skeletal defects and facial dysmorphism (usually includes hypertelorism and down slanting palpebral fissures). PTPN11 hot spot exon testing identifies mutations in more than half of Noonan syndrome patients.

Contributors: ML: study design, article writing, data collection; ICV: article review, critical input, study design, data collection; RDP: article critical review and writing, data collection, study design; SBM: article critical review, data collection; KM: article critical review, data collection, PTPN11 test. All authors approved the final version of manuscript.

Funding; None; Competing interest; None stated.