title: Highly sensitive diagnosis of 43 monogenic forms of diabetes or obesity through one-step PCR-based enrichment in combination with next-generation sequencing.
abstract: 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.
  
  
  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.
authors.affiliation: Corresponding author: Philippe Froguel, p.froguel@imperial.ac.uk.
authors.forename: Amélie
authors.initials: A
authors.lastname: Bonnefond
authors.forename: Julien
authors.initials: J
authors.lastname: Philippe
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Durand
authors.forename: Jean
authors.initials: J
authors.lastname: Muller
authors.forename: Sadia
authors.initials: S
authors.lastname: Saeed
authors.forename: Muhammad
authors.initials: M
authors.lastname: Arslan
authors.forename: Rosa
authors.initials: R
authors.lastname: Martínez
authors.forename: Franck
authors.initials: F
authors.lastname: De Graeve
authors.forename: Véronique
authors.initials: V
authors.lastname: Dhennin
authors.forename: Iandry
authors.initials: I
authors.lastname: Rabearivelo
authors.forename: Michel
authors.initials: M
authors.lastname: Polak
authors.forename: Hélène
authors.initials: H
authors.lastname: Cavé
authors.forename: Luis
authors.initials: L
authors.lastname: Castaño
authors.forename: Martine
authors.initials: M
authors.lastname: Vaxillaire
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Mandel
authors.forename: Olivier
authors.initials: O
authors.lastname: Sand
authors.forename: Philippe
authors.initials: P
authors.lastname: Froguel
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D003362:Cost-Benefit Analysis; D003920:Diabetes Mellitus / Q000175:diagnosis* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D009154:Mutation; D009765:Obesity / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity