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Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia

Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial...

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Autores principales: Aloi, Concetta, Salina, Alessandro, Caroli, Francesco, Bocciardi, Renata, Tappino, Barbara, Bassi, Marta, Minuto, Nicola, d’Annunzio, Giuseppe, Maghnie, Mohamad
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10221606/
https://www.ncbi.nlm.nih.gov/pubmed/37240725
http://dx.doi.org/10.3390/life13051080
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author Aloi, Concetta
Salina, Alessandro
Caroli, Francesco
Bocciardi, Renata
Tappino, Barbara
Bassi, Marta
Minuto, Nicola
d’Annunzio, Giuseppe
Maghnie, Mohamad
author_facet Aloi, Concetta
Salina, Alessandro
Caroli, Francesco
Bocciardi, Renata
Tappino, Barbara
Bassi, Marta
Minuto, Nicola
d’Annunzio, Giuseppe
Maghnie, Mohamad
author_sort Aloi, Concetta
collection PubMed
description Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants.
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spelling pubmed-102216062023-05-28 Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia Aloi, Concetta Salina, Alessandro Caroli, Francesco Bocciardi, Renata Tappino, Barbara Bassi, Marta Minuto, Nicola d’Annunzio, Giuseppe Maghnie, Mohamad Life (Basel) Brief Report Next-generation sequencing (NGS) has revolutionized the field of genomics and created new opportunities for basic research. We described the strategy for the NGS validation of the “dysglycaemia panel” composed by 44 genes related to glucose metabolism disorders (MODY, Wolfram syndrome) and familial renal glycosuria using Ion AmpliSeq technology combined with Ion-PGM. Anonymized DNA of 32 previously genotyped cases with 33 different variants were used to optimize the methodology. Standard protocol was used to generate the primer design, library, template preparation, and sequencing. Ion Reporter tool was used for data analysis. In all the runs, the mean coverage was over 200×. Twenty-nine out of thirty three variants (96.5%) were detected; four frameshift variants were missed. All point mutations were detected with high sensitivity. We identified three further variants of unknown significance in addition to pathogenic mutations previously identified by Sanger sequencing. The NGS panel allowed us to identify pathogenic variants in multiple genes in a short time. This could help to identify several defects in children and young adults that have to receive the genetic diagnosis necessary for optimal treatment. In order not to lose any pathogenic variants, Sanger sequencing is included in our analytical protocol to avoid missing frameshift variants. MDPI 2023-04-24 /pmc/articles/PMC10221606/ /pubmed/37240725 http://dx.doi.org/10.3390/life13051080 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Brief Report
Aloi, Concetta
Salina, Alessandro
Caroli, Francesco
Bocciardi, Renata
Tappino, Barbara
Bassi, Marta
Minuto, Nicola
d’Annunzio, Giuseppe
Maghnie, Mohamad
Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
title Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
title_full Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
title_fullStr Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
title_full_unstemmed Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
title_short Next Generation Sequencing (NGS) Target Approach for Undiagnosed Dysglycaemia
title_sort next generation sequencing (ngs) target approach for undiagnosed dysglycaemia
topic Brief Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10221606/
https://www.ncbi.nlm.nih.gov/pubmed/37240725
http://dx.doi.org/10.3390/life13051080
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