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 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 200x. 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.

Automated summary

Next-generation sequencing (NGS) has revolutionized genomics and provided new opportunities for basic research. We used Ion AmpliSeq technology combined with Ion-PGM to validate a dysglycaemia panel of 44 genes related to glucose metabolism disorders. By optimizing the methodology with previously genotyped samples, we achieved high sensitivity in detecting point mutations and identified additional variants of unknown significance. The NGS panel enabled us to quickly identify pathogenic variants in multiple genes, which is crucial for optimal treatment in children and young adults.