Diagnosis of Noonan syndrome and related disorders using target next generation sequencing.
BACKGROUND
Noonan syndrome is an autosomal dominant developmental disorder with a high phenotypic variability, which shares clinical features with other rare conditions, including LEOPARD syndrome, cardiofaciocutaneous syndrome, Noonan-like syndrome with loose anagen hair, and Costello syndrome. This group of related disorders, so-called RASopathies, is caused by germline mutations in distinct genes encoding for components of the RAS-MAPK signalling pathway. Due to high number of genes associated with these disorders, standard diagnostic testing requires expensive and time consuming approaches using Sanger sequencing. In this study we show how targeted Next Generation Sequencing (NGS) technique can enable accurate, faster and cost-effective diagnosis of RASopathies.


METHODS
In this study we used a validation set of 10 patients (6 positive controls previously characterized by Sanger-sequencing and 4 negative controls) to assess the analytical sensitivity and specificity of the targeted NGS. As second step, a training set of 80 enrolled patients with a clinical suspect of RASopathies has been tested. Targeted NGS has been successfully applied over 92% of the regions of interest, including exons for the following genes: PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC, MAP2K1, MAP2K2, CBL.


RESULTS
All expected variants in patients belonging to the validation set have been identified by targeted NGS providing a detection rate of 100%. Furthermore, all the newly detected mutations in patients from the training set have been confirmed by Sanger sequencing. Absence of any false negative event has been excluded by testing some of the negative patients, randomly selected, with Sanger sequencing.


CONCLUSIONS
Here we show how molecular testing of RASopathies by targeted NGS could allow an early and accurate diagnosis for all enrolled patients, enabling a prompt diagnosis especially for those patients with mild, non-specific or atypical features, in whom the detection of the causative mutation usually requires prolonged diagnostic timings when using standard routine. This approach strongly improved genetic counselling and clinical management.
Cytogenetics, Medical Genetics and Pediatric Cardiology, Bambino Gesù Children Hospital, IRCCS, Rome, Italy. francescaromana.lepri@opbg.net.
Francesca Romana
FR
Lepri
Rossana
R
Scavelli
Maria Cristina
MC
Digilio
Maria
M
Gnazzo
Simona
S
Grotta
Maria Lisa
ML
Dentici
Elisa
E
Pisaneschi
Pietro
P
Sirleto
Rossella
R
Capolino
Anwar
A
Baban
Serena
S
Russo
Tiziana
T
Franchin
Adriano
A
Angioni
Bruno
B
Dallapiccola
D000293:Adolescent; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007223:Infant; D008297:Male; D009154:Mutation; D009634:Noonan Syndrome / Q000175:diagnosis* / Q000235:genetics*; D015203:Reproducibility of Results