Highly Sensitive Diagnosis of 43 Monogenic Forms of Diabetes or Obesity Through One-Step PCR-Based Enrichment in Combination With Next-Generation Sequencing

• DOI: 10.2337/dc13-0698
• Publication Date: 2013-09-17T04:35:56Z
• Authors: Amélie Bonnefond; Julien Philippe; Emmanuelle Durand; Jean Muller; Sadia Saeed; Muhammad Arslan; Rosa Martínez; Franck De Graeve; Véronique Dhennin; Iandry Rabearivelo; Michel Polak; Hélène Cavé; Luis Castaño; Martine Vaxillaire; Jean-Louis Mandel; Olivier Sand; Philippe Froguel
• Keywords: 2. Zero hunger; 0303 health sciences; Adolescent; Cost-Benefit Analysis; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Sensitivity and Specificity; 3. Good health; 03 medical and health sciences; Child, Preschool; Mutation; Diabetes Mellitus; Humans; [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology; Obesity; Child

OBJECTIVE Accurate etiological diagnosis of monogenic forms of diabetes and obesity is useful as it can lead to marked improvements in patient care and genetic counseling. Currently, molecular diagnosis based on Sanger sequencing is restricted to only a few genes, as this technology is expensive, time-consuming, and labor-intensive. High-throughput next-generation sequencing (NGS) provides an opportunity to develop innovative cost-efficient methods for sensitive diabetes and obesity multigene screening.

RESEARCH DESIGN AND METHODS We assessed a new method based on PCR enrichment in microdroplets (RainDance Technologies) and NGS using the Illumina HiSeq2000 for the molecular diagnosis of 43 forms of monogenic diabetes or obesity. Forty patients carrying a known causal mutation for those subtypes according to diagnostic laboratories were blindly reanalyzed.

RESULTS Except for one variant, we reidentified all causal mutations in each patient associated with an almost-perfect sequencing of the targets (mean of 98.6%). We failed to call one highly complex indel, although we identified a dramatic drop of coverage at this locus. In three patients, we detected other mutations with a putatively deleterious effect in addition to those reported by the genetic diagnostic laboratories.

CONCLUSIONS Our NGS approach provides an efficient means of highly sensitive screening for mutations in genes associated with monogenic forms of diabetes and obesity. As cost and time to deliver results have been key barriers to uncovering a molecular cause in the many undiagnosed cases likely to exist, the present methodology should be considered in patients displaying features of monogenic diabetes or obesity.