Supplemental data from: Next-generation sequencing base calls for mosaic mutations

Context: Some children with diazoxide-unresponsive congenital hyperinsulinism (HI) lack any detectable disease-causing mutation in peripheral blood DNA. 

Objective: To examine whether somatic post-zygotic mutations of known HI genes are responsible for disease in children with diazoxide-unresponsive HI requiring surgery with histology not classified as focal or LINE, and without detectable mutations by standard genetic testing of peripheral blood DNA. 

Methods: Next-generation sequencing (NGS) was performed on specimens of pancreas from 10 children with diazoxide-unresponsive HI.

Results: Four unique GCK mutations were identified at low levels of mosaicism ranging from 4.4-10.1% in pancreatic DNA from five of these 10 children.  The GCK mutations were not detectable in peripheral blood DNA by NGS in three cases from which peripheral blood DNA was available for testing.  All four GCK mutations have been previously published as activating HI mutations.     

Conclusion: These results indicate that post-zygotic somatic mutations of known HI genes are responsible for some cases of diazoxide-unresponsive hyperinsulinism.

Methods
Next Generation Sequencing:  Pancreatic islets were isolated from surgical specimens as previously described (20,21). DNA and RNA were isolated from peripheral blood and pancreatic tissue using standard kits following manufacturer’s instructions.  Exonic and flanking intronic regions of ABCC8, KCNJ11, and GCK, were PCR amplified and combined in equimolar concentrations prior to NGS (Ion Torrent, Life Technologies) as previously described (14).  In order to detect low-level mosaic mutations, NGS was performed with a high depth of coverage.  The average depth of coverage for the cases included here was 17,612x compared to ~100x that is typically offered on standard clinical testing (22). Signal processing and base calling were performed using the Torrent Suite Software, followed by alignment with the Torrent Mapping Alignment Program (TMAP). Variant calling and coverage analysis were performed on aligned reads using the Torrent Variant Caller and Coverage Analysis plugins (Life Technologies, Carlsbad, CA). Variant calling included a low stringency filter to identify mosaic variants as low as 2% using the “somatic low stringency” configuration in the Torrent Variant Caller plugin.

GCK sequence information is based on GenBank reference sequence NM_000162.3.  Variants were searched against the gnomAD Browser (v2.1) (23).  Variants were confirmed by direct sequencing as previously described (1).