Seventeen patients with clinical diagnosis of Noonan syndrome, based on Van der Burgt criteria [9] were included in the study. Ethical clearance was obtained from the Institute Ethics Committee. Informed consent was obtained from the patient or the parents in case of minors, for storage of blood and mutation analysis. Physical characteristics were noted and anthropometry was done in all patients. All patients underwent echocardiography for congenital heart disease. Karyotype was done in all subjects to rule out chromosomal disorders. Sanger sequencing of all the 15 exons of PTPN11was done. A genotype–phenotype correlation was attempted by comparing the clinical features of patients with and without mutation in PTPN11.

The age of presentation ranged from 2 months to 18 years (11 males). Table I shows the clinical features present in comparison to the mutation identified in exon 3 and exon 13 of PTPN11. Table II shows the clinical features and the mutations identified in individual patients. The typical facial features of Noonan syndrome in subjects with a mutation identified in PTPN11are given in Web Fig.1. Of the facial features, down slanting eyes was the consistent feature, which was present in all patients. All the patients in our cohort had short neck and short stature. The height in the study cohort ranged from -2 to -4 SD below mean.

TABLE I	Association of Clinical Features and the Mutations in PTPN 11 in the Study Children

TABLE II Clinical Features and Mutation Spectrum of Probands

Cardiovascular abnormalities were present in 16 (94%); the most common abnormality being pulmonary stenosis(62.5%). Echocardiography was normal in only one patient with PTPN11 mutation.

Mild cognitive impairment was present in 6 (35.2%) patients; 5 of them had a mutation in PTPN11.Of the 17 probands, 4 (25%) had other affected family members. Mutation in PTPN11 was detected in 64.7% of patients; 8 (72%) had mutation in exon 3 and three patients had mutation in exon 13 (27%). All the variants were previously reported disease - causing variants and were reported in Human Gene Mutation Database.

Noonan syndrome is an autosomal dominant disorder with short stature, facial dysmorphism and congenital heart diseases[10]. In 20-30% of cases, disease-causing variants have not yet been identified [10].

Congenital heart disease is seen in more than 90% of Noonan syndrome, with pulmonary stenosis being the most common defect [5,10]. This finding is replicated in our study where 94% had congenital heart disease and the most common abnormality noted was pulmonary stenosis.PTPN11 accounts for 50% of all cases of Noonan syndrome and is more prevalent in individuals with short stature and pulmonary stenosis [5]. PTPN11 mutations have also been linked to easy bruising, pectus deformity and characteristic facial appearance [11]. In our cohort, all patients with PTPN11 mutation had short stature, short neck and down slanting eyes. Out of these 11 patients, six (54%) had pulmonary stenosis, consistent with previous reports of pulmonary stenosis being more common in PTPN11 mutation. As with previous studies, pulmonary stenosis remains as a marker in predicting mutation in PTPN11[5].

Exon 3 and exon 8 in PTPN11 were identified as mutation hotspots in previous studies [5,12]. In our study, heterozygous variants were seen in exon 3 in 8 out of 11 individuals, comparable with previous reports. We did not identify any variant in exon 8, but variants were found in exon 13 in two individuals. Since the sample size was limited we could not draw any definite phenotypic correlation with the exon in which mutation was identified.

We propose that exon 3 and exon 13 screening should be done in Indian subjects with short stature, downslanting eyes, short neck and pulmonary stenosis as a first step, followed by screening of other exons for variants. If no PTPN11 mutation is identified, this should be followed by panel testing for the other genes like SOS1, RAF1, KRAS, NRAS, SHOC2, CBL and RIT1. Other differential diagnosis includes conditions like Costello syndrome, cardiofaciocutaneous syndrome, LEOPARD syndrome etc. Early identification and multi- disciplinary management is essential for better outcome- among this group of patients. Identification of disease causing variant in a family is essential in providing prenatal diagnosis.

Contributors: DLN, HP, AM, RG, RGP, SJP: substantial contributions to the design of the work; acquisition, analysis, and interpretation of data; drafting the manuscript; approval of version to be published; and accountable for all aspects of the work. KM, SRP: substantial contributions to the design of the work, acquisition, analysis, and interpretation of data for the work, has revised the manuscript critically and given suggestions and has approved version to be published and agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding: SGPGIMS Intramural 2014-63-IMP-76;

Competing interests: None stated.

1. Noonan JA. Hypertelorism with Turner phenotype. A new syndrome with associated congenital heart disease. Am J Dis Child.1968;16:373-80.

2. Nora JJ, Nora AH, Sinha AK, Spangler RD, Lubs HA. The Ullrich-Noonan syndrome (Turner phenotype). Am J Dis Child. 1974;127:48-55.

3. Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H, et al. Mutations in PTPN11, encoding the protein tyrosine phosphatise SHP-2, cause Noonan syndrome. Nat Genet. 2001;29: 465-8.

4. Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, van der Burgt I, et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet. 2002; 70:1555-63.

5. Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, Joshi VA, et al. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet. 2007;39:70-4.

6. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, et al. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet. 2007;39:1007-12.

7. Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, et al.Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet. 2007;39:1013.

8. Aoki Y, Niihori T, Banjo T, Okamoto N, Mizuno S, Kurosawa K, et al. Gain-of function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome. Am J Hum Genet. 2013; 93:173-80 .

9. Van der Burgt I. Noonan syndrome. Orphanet J Rare Dis. 2007;2:4.

10. Romano AA, Allanson JE, Dahlgren J, Gelb BD, Hall B, Pierpont ME, et al. Noonan syndrome: Clinical features, diagnosis, and management guidelines. Pediatrics.2010; 26:746-59.

11. Tartaglia M, Gelb BD, Zenker M. Noonan syndrome and clinically related disorders. Best Pract Res Clin Endocrinol Metab. 2011;25:161-79.