Diagnosis of Noonan syndrome and related disorders using target next generation sequencing

Francesca R. Lepri, Rossana Scavelli, Maria C. Digilio, Maria Gnazzo, Simona Grotta, Maria L. Dentici, Elisa Pisaneschi, Pietro Sirleto, Rossella Capolino, Anwar Baban, Serena Russo, Tiziana Franchin, Adriano Angioni, Bruno Dallapiccola

Research output: Contribution to journalArticle

Abstract

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. Conclusion: 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.

Original languageEnglish
Article number14
JournalBMC Medical Genetics
Volume15
Issue number1
DOIs
Publication statusPublished - Jan 23 2014

Fingerprint

Noonan Syndrome
Loose Anagen Hair Syndrome
Costello Syndrome
LEOPARD Syndrome
Gene Components
Mutation
Germ-Line Mutation
Genetic Counseling
Genes
Early Diagnosis
Exons
Costs and Cost Analysis
Sensitivity and Specificity

Keywords

  • Molecular diagnosis
  • Next generation sequencing
  • Noonan syndrome
  • RASopathies

ASJC Scopus subject areas

  • Genetics(clinical)
  • Genetics

Cite this

@article{b0ffa7e9dafe46a1ae2011fb836a61a5,
title = "Diagnosis of Noonan syndrome and related disorders using target next generation sequencing",
abstract = "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. Conclusion: 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.",
keywords = "Molecular diagnosis, Next generation sequencing, Noonan syndrome, RASopathies",
author = "Lepri, {Francesca R.} and Rossana Scavelli and Digilio, {Maria C.} and Maria Gnazzo and Simona Grotta and Dentici, {Maria L.} and Elisa Pisaneschi and Pietro Sirleto and Rossella Capolino and Anwar Baban and Serena Russo and Tiziana Franchin and Adriano Angioni and Bruno Dallapiccola",
year = "2014",
month = "1",
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language = "English",
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journal = "BMC Medical Genetics",
issn = "1471-2350",
publisher = "BioMed Central",
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TY - JOUR

T1 - Diagnosis of Noonan syndrome and related disorders using target next generation sequencing

AU - Lepri, Francesca R.

AU - Scavelli, Rossana

AU - Digilio, Maria C.

AU - Gnazzo, Maria

AU - Grotta, Simona

AU - Dentici, Maria L.

AU - Pisaneschi, Elisa

AU - Sirleto, Pietro

AU - Capolino, Rossella

AU - Baban, Anwar

AU - Russo, Serena

AU - Franchin, Tiziana

AU - Angioni, Adriano

AU - Dallapiccola, Bruno

PY - 2014/1/23

Y1 - 2014/1/23

N2 - 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. Conclusion: 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.

AB - 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. Conclusion: 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.

KW - Molecular diagnosis

KW - Next generation sequencing

KW - Noonan syndrome

KW - RASopathies

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