Low-level mosaic variants causing the pancreatic disease congenital hyperinsulinism can be detected from blood DNA
Jasmin J Bennett1, Thomas W Laver1, Jonna M E Männistö2
1Clinical and Biomedical Science, University of Exeter, Exeter, UK.
Background:
A substantial proportion of individuals with a well-defined monogenic disorder remain without a genetic diagnosis. Low-level mosaic pathogenic variants are recognised as an underappreciated cause of monogenic disease but are technically challenging to detect, particularly in organ-specific conditions when affected tissue is inaccessible.
Methods:
We systematically investigated low-level mosaic variants in individuals with congenital hyperinsulinism (CHI: n = 1252) or neonatal diabetes (NDM: n = 312), two opposing pancreatic disorders of insulin secretion. We screened for established pathogenic variants with variant allele fraction (VAF) < 8% in dominant CHI (ABCC8, GCK, GLUD1, HK1) or dominant NDM (ABCC8, KCNJ11, INS) genes in targeted next-generation sequencing (tNGS) data using Mutect2.
Findings:
This called 40 variants across the four genes in 39 individuals with CHI. No candidate variants were found in the NDM cohort. Orthogonal validation of 35 variants using TaqMan-based droplet digital PCR (ddPCR) confirmed 26/35 variants. The median VAF for confirmed variants was 3.6% (1.0-7.8%), while false positives (9/35) predominantly had a VAF <1% with some overlap in VAF with true positives.
Interpretation:
This study shows that disease-causing low-level mosaic variants in dominant CHI genes can be detected in blood using tNGS but require orthogonal validation. These results provide a framework to improve diagnostic yield in organ-specific conditions where mosaic variants may represent an important missed cause of disease.
Funding:
This work was supported by a research grant from the University of Pennsylvania Orphan Disease Center in partnership with the Team CHIbra and Congenital Hyperinsulinism International [MDBR-23-020-CHI] and the Wellcome Trust [223187/Z/21/Z].
Insights
Low-level mosaic variants in congenital hyperinsulinism (CHI) genes can be detected in blood using targeted next-generation sequencing (tNGS). Orthogonal validation is crucial for confirming these low-frequency pathogenic variants.
Area of Science:
- Genetics
- Molecular Biology
- Pediatrics
Background:
- Many individuals with monogenic disorders lack a genetic diagnosis.
- Low-level mosaic pathogenic variants are a significant, yet underdiagnosed, cause of genetic disease, especially in organ-specific conditions.
- Detecting these variants is challenging due to their low variant allele fraction (VAF).
Purpose of the Study:
- To investigate the utility of targeted next-generation sequencing (tNGS) for detecting low-level mosaic variants in congenital hyperinsulinism (CHI) and neonatal diabetes (NDM).
- To assess the diagnostic yield of tNGS in identifying pathogenic variants with VAF < 8% in these pancreatic disorders.
Main Methods:
- Systematic screening of targeted next-generation sequencing (tNGS) data for low-level mosaic variants (VAF < 8%) in individuals with CHI (n=1252) and NDM (n=312).
- Analysis focused on dominant genes associated with CHI (ABCC8, GCK, GLUD1, HK1) and NDM (ABCC8, KCNJ11, INS).
- Orthogonal validation of candidate variants using TaqMan-based droplet digital PCR (ddPCR).
Main Results:
- Forty variants were identified across four CHI genes in 39 individuals; no variants were found in the NDM cohort.
- Orthogonal validation confirmed 26 out of 35 variants, with a median VAF of 3.6% for true positives.
- False positives (9/35) typically had VAF < 1%, indicating the need for careful interpretation and validation.
Conclusions:
- Targeted next-generation sequencing (tNGS) can detect disease-causing low-level mosaic variants in congenital hyperinsulinism (CHI) genes from blood samples.
- Orthogonal validation is essential to confirm true positive findings and differentiate from false positives.
- This approach provides a framework to improve diagnostic yield for organ-specific genetic conditions where mosaic variants are often missed.
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