- Diabetes Genes
- Tests for Diabetes Subtypes
- Targeted Next Generation Sequencing
Targeted Next Generation Sequencing
Analysis of 71 Monogenic Diabetes Genes
The Exeter Molecular Genetics Laboratory offers a customised, targeted next generation sequencing capture assay for variant analysis of 71 known/putative monogenic diabetes genes and 6 mitochondrial DNA (mtDNA) variants associated with diabetes. Analysis of a sub-set of genes is performed for paediatric and early adult onset diabetes (including MODY and insulin resistance disorders), neonatal diabetes and autoimmune forms of neonatal diabetes.
For patients with paediatric and early adult onset diabetes a panel of 39 genes is sequenced that includes 11 MODY genes (ABCC8, APPL1, GCK, HNF1A, HNF4A, INS, KCNJ11, NEUROD1, PDX1, RFX6 and ZFP57), 19 genes where diabetes occurs as part of a syndrome (CEL, CISD2, DCAF17, DNAJC3, DUT, DYRK1B, GATA4, GATA6, HNF1B, MANF, MAFA, PAX6, PCBD1, PIK3R1, PPP1R15B, SLC29A3, TRMT10A, WFS1, ZBTB20), 9 genes where pathogenic variants cause diabetes through severe insulin resistance with or without partial lipodystrophy (AKT2, CIDEC, INSR, LIPE, LMNA, PLIN1, POLD1, PPARG and ZMPSTE24) and the mtDNA variants m.3243A>G, m.8344A>G, m.12258C>A, m.12271T>C, m.14684C>T and m.14709T>C causing mitochondrial diabetes.
A panel of 35 genes is sequenced for patients diagnosed with neonatal diabetes under the age of 6 months (ABCC8, AGPAT2, BSCL2, CISD2, CNOT1, COQ2, COQ9, EIF2B1, EIF2S3, EIF2AK3, FOXP3, GATA4, GATA6, GCK, GLIS3, HNF1B, IER3IP1, IL2RA, INS, INSR, KCNJ11, LPL, LRBA, MNX1, NEUROD1, NEUROG3, NKX2-2, PDX1, PTF1A (coding and distal enhancer regions), RFX6, SLC19A2, SLC2A2, STAT3, WFS1 and ZFP57) and a panel of 15 genes is sequenced for patients with neonatal diabetes and autoimmune disease (AIRE, CTLA4, DOCK8, FOXP3, IL2RA, ITCH, JAK1, LRBA, NFKB1, SIRT1, SLC29A3, STAT1, STAT3, STAT5B and TNFAIP3).
The targeted next generation sequencing assay “captures” the protein coding regions and conserved splice sites of the 71 monogenic diabetes genes and the regions of the mitochondrial genome containing the 6 mtDNA variants from the patient DNA samples by hybridisation. These DNA fragments are then amplified and sequenced on an Illumina NextSeq 500 or NextSeq 550 to generate multiple sequence reads per base.
5-10ml venous blood in plastic EDTA bottles or >5ug DNA
- Variant analysis of the genes listed above is carried out using a custom Twist Bioscience NGS target enrichment system and the Illumina NextSeq 500 or NextSeq 550 next generation sequencing platforms by:
- Sequence analysis of the coding regions and intronic regions located within 100 bp upstream and 60 bp downstream of each exon using 150bp paired end reads
- Dosage analysis for deletions, insertions and duplications of ≥30 nucleotides by relative read depth coverage using the ExomeDepth tool (Parrish et al 2017 Wellcome Open Res 2: 49)
- 99.4% of the analysed bases from 20 genes currently tested by Sanger sequencing are covered by a minimum of 30 reads (98.5% bases with ≥30 reads for all 71 genes). Low coverage of GATA6 exon 2 will be supplemented by Sanger sequencing for any patients with a cardiac malformation.
- More than 4000 samples have been tested to date. A validation series of 70 known single nucleotide variants/indels/exonic deletions or duplications demonstrated 100% sensitivity and specificity (Ellard et al 2013 Diabetologia 56: 1958-1963, open access available at http://dx.doi.org/10.1007/s00125-013-2962-5).
- Confirmation of complex insertion/deletion/duplication (indel) variants identified by tNGS is undertaken by Sanger sequencing for the purposes of ensuring that the correct nucleotide change is reported. Simple indels will be confirmed by visual inspection of mapped sequence reads using IGV and will not be confirmed by Sanger sequencing unless indicated otherwise (e.g. located in a region of repetitive DNA sequence or if evidence-based quality parameters are not met). Dosage analysis by MLPA or droplet digital PCR is performed to confirm larger partial and whole gene deletions (excluding the HNF1B gene deletion since no false positive deletions have been reported by this assay to date). Single nucleotide substitutions that meet evidence-based quality parameters will not be confirmed by Sanger sequencing. No false positive results have been reported by this assay to date.