Providing information for patients and professionals on research and clinical care in genetic types of diabetes.

Targeted Next Generation Sequencing Analysis of 45 Monogenic Diabetes Genes

The Exeter Molecular Genetics Laboratory offers a customised, targeted next generation sequencing capture assay for the mutation analysis of 45 known/putative monogenic diabetes genes and the m.3243 region of the mitochondrial genome.  Analysis of a sub-set of genes is performed for MODY, neonatal diabetes and autoimmune forms of monogenic diabetes. 

For patients with paediatric and early adult onset diabetes a panel of 24 genes is sequenced that includes the MODY genes ABCC8, CEL, CISD2, GATA4, GATA6, GCK, HNF1A, HNF1B, HNF4A, INS, KCNJ11, NEUROD1, PAX6, PCBD1, PDX1, RFX6, TRMT10A, WFS1 and ZFP57, five genes where mutations cause diabetes through severe insulin resistance (LMNA, PPARG, PLIN1, INSR and POLD1) and the mitochondrial mutation m.3243A>G causing maternally inherited diabetes and deafness (MIDD).

A panel of 28 genes is sequenced for patients diagnosed with neonatal diabetes under the age of 6 months (ABCC8, BSCL2, CISD2, EIF2AK3, FOXP3, GATA4, GATA6, GCK, GLIS3, HNF1B, IER3IP1, IL2RA, INS, INSR, LRBA, KCNJ11, MNX1 (exon 2 only), NEUROD1, NEUROG3, NKX2-2, PDX1, PTF1A (coding and distal enhancer regions), RFX6, SLC19A2, SLC2A2, STAT3, WFS1 and ZFP57) and a panel of 8 genes is sequenced for patients with neonatal diabetes and autoimmune disease (FOXP3, IL2RA, ITCH, LRBA, SIRT1, STAT1, STAT3 and STAT5).

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The targeted next generation sequencing assay “captures” the protein coding regions and conserved splice sites of the 28 monogenic diabetes genes and the m.3243 region of the mitochondrial genome from the patient DNA samples by hybridisation. These DNA fragments are then amplified and sequenced on an Illumina HiSeq 2000/2500 to generate multiple sequence reads per base.


5-10ml venous blood in plastic EDTA bottles or >5ug DNA


  • Mutation analysis of the genes listed above is carried out using a custom Agilent SureSelect system and the Illumina NextSeq 500 next generation sequencing platform by:


  • Sequence analysis of the coding regions and intronic regions located within 50 bp upstream and 10 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 (Plagnol et al 2012 Bioinformatics 28: 2747-2754)


  • 99.4% of the analysed bases from 20 genes currently tested by Sanger sequencing are covered by a minimum of 30 reads (97.5% bases with ≥30 reads for all 45 genes). Low coverage of GATA6 exon 2 will be supplemented by Sanger sequencing for any patients with a cardiac malformation.


  • More than 1700 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  


  • Confirmation of mutations identified by tNGS is undertaken by Sanger sequencing for single nucleotide substitutions and small indels, dosage analysis by MLPA for larger partial and whole gene deletions and TaqMan real-time PCR for the mitochondrial mutation m.3243A>G.