We designed a custom Agilent SureSelect exon-capture assay (Agilent Technologies, Santa Clara, CA, USA) with baits for 29 genes (see electronic supplementary material [ESM] Methods). These included 13 known/putative MODY genes (GCK, HNF1A, HNF4A, HNF1B, NEUROD1, INS, CEL, PDX1, PAX4, BLK, KLF11, KCNJ11 and ABCC8), two genes where mutations cause diabetes through lipodystrophy (LMNA and PPARG), the m.3243 region of the mitochondrial genome (where the m.3243A>G mutation causes MIDD) and 20 neonatal diabetes genes (GCK, KCNJ11, ABCC8, INS, PDX1, PTF1A, HNF1B, NEUROD1, NEUROG3, RFX6, EIF2AK3, FOXP3, GLIS3, SLC19A2, SLC2A2, IER3IP1, ZFP57, WFS1, GATA6 and GATA4). Bait density and replication were adjusted using coverage data from exome sequencing samples, captured with the Agilent SureSelect Human All Exon v1 (38 Mb) system (see ESM Fig. 1), to achieve greater uniformity of capture across the 66.8 kb target.
DNA samples from a total of 114 patients were tested—32 with known mutations identified by Sanger sequencing, MLPA dosage or array CGH (comparative genomic hybridisation) and 82 previously tested for MODY (n = 33, diagnosed at age <35 years, BMI <30 kg/m2, who had undergone previous testing for GCK, HNF1A and/or HNF4A) or neonatal diabetes diagnosed before 6 months (n = 49, all previously tested for mutations in at least KCNJ11, ABCC8 and INS) but in whom a mutation had not been found. Study participants gave informed consent and these investigations were carried out in accordance with the Declaration of Helsinki as revised in 2000.
Samples were fragmented using a Bioruptor (Diagenode, Liège, Belgium), indexed for multiplexing and hybridised (in pools of 12 samples) according to the manufacturer’s instructions. Sequencing was performed with an Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) (48 samples per lane) and 100 bp paired end reads. Data were processed as described previously  to identify potential pathogenic mutations located within 50 bp upstream and 10 bp downstream of each exon. Deletions/duplications >30 bp were identified by relative read depth coverage. All newly identified mutations were confirmed by Sanger sequencing.