Improved genetic testing for monogenic diabetes using targeted next-generation sequencing

Abstract

Aims/hypothesis: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes.

Methods: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested--32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications.

Results: In the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested.

Conclusions/interpretation: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate.

Figures

Fig. 1

Depth of coverage for targeted genes. (a) The percentage of targeted bases sequenced at a minimum depth of 2, 10, 20 and 30 reads per base for regions of interest for all 28 MODY/neonatal diabetes genes and the m.3243 base (black bars) and for the 20 genes routinely tested by Sanger sequencing in our laboratory (white bars). Data are mean values for the cohort of 114 samples; error bars show 1 SD from the mean. (b) Region of low coverage (<30 reads per base) within GATA6 exon 2. The graph shows the average depth of coverage (black line) in the region chr18:19,751,740–19,752,040 (hg19 coordinates); grey shading indicates 1 SD from the mean. Average coverage depth over the entire GATA6 exon 2 region of interest (chr18:19,751,056–19,752,250) was 126