Highly sensitive diagnosis of 43 monogenic forms of diabetes or obesity, through one step PCR-based enrichment in combination with next generation sequencingJulien Philippe1,2,3,*, Amélie Bonnefond1,2,3, Emmanuelle Durand1,2,3, Jean Muller4,5, Sadia Saeed6, Muhammad Arslan7,

Rosa M. Martínez Salazar8,9, Franck De Graeve1,2,3, Véronique Dhennin1,2,3, Iandry Rabearivelo1,2,3, Michel Polak10,11, Hélène Cavé12, Luis Castaño7,8, Martine Vaxillaire1,2,3, Jean-Louis Mandel4,5,13, Olivier Sand1,2,3 & Philippe Froguel1,2,3,6

1European Genomic Institute for Diabetes (EGID), Lille, France; 2CNRS UMR8199, Pasteur Institute of Lille, Lille, France; 3Lille 2 University, Lille, France; 4Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS UMR7104, Inserm U964, Université de Strasbourg, Illkirch, France; 5Laboratoire de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 6Department of Genomics of Common Disease, Hammersmith Hospital, Imperial College London, London, UK; 7Centre for Research in Endocrinology and Reproductive Sciences, University of Health Sciences, Lahore, Pakistan; 8Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Bilbao, Spain; 9Research Unit, Cruces University Hospital, UPV/EHU, Barakaldo, Spain; 10Inserm U845, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; 11Department of Paediatric Endocrinology, Necker Enfants-Malades Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; 12Department of Genetics, Robert-Debré Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; 13Chaire de Génétique Humaine, Collège de France, Illkirch, France.

* Email : julien@good.ibl.fr

Objectives•Todevelopacost-effectivehigh-throughputapproachformoleculardiagnosisofgeneticdiseasesthatischeaperandquickerthanWES,andless

labor-intensive than the gold standard Sanger sequencing•To assess a new technology in 40 patients presenting monogenic forms of diabetes or obesity, of which causal mutations were already known

MethodsWedesignedaPCRbasedsequenceenrichmentpaneltargetingtheexonsof43knownsusceptibilitygenes for monogenic forms of diabetes and obesity. This panel included a total of 970 primer pairs targeting a total of 437kb of sequence.

Each of the 40 samples genomic DNA was processed on the RDT1000 (RainDance). Fragmented gDNA mixed with PCR reagents was processed into microdroplets that were subsequently fused with microdropletscontainingtheprimerlibrary.Theresultingmicrodropletsweresubsequentlyamplifiedby PCR. Finally, all 40 amplicon enriched samples were sequenced on the HiSeq2000 (Illumina) to a minimum coverage of 100×.

Results

Conclusion

40 patients

1 patient not confirmed

1 missed indel in BBS5: c.572_594inv{ins567_56

8};595_603del

39 patients

confirmed

3 patients with several potentially causal mutations

1 neonatal diabetes with mutation in

ABCC8 & KCNJ11

1 BBS patient with a mutation in ABCC8

1 obese patient with a mutation in ABCC8

32 punctual mutations and 7

deletions [1 à 5pb]

Indel not detected by our bioinformatics pipeline

Diabetes Obesity

Neonatal & syndromic Neonatal MODY Syndromic Nonsyndromic

FOXP3 PDX1 POMCCEL ABCC8 BBS1-16 LEP

WFS1 GCK ALMS1 LEPREIF2AK3 INS GNAS BDNF

NEUROG3 KCNJ11 MC4RRFX6 NEUROD1 SIM1

PTF1A HNF1A NTRK2GLIS3 HNF1B PCSK1

HNF4A

Raindance technology Sanger sequencing

Pros (+)• High throughput (48 samples/24 hours)• High coverage (20000 amplicons)• PCR without allelic bias

• Low error rate•Very high quality / base• Gold standard

Cons (-)• Complex bioinformatics analysis• NGS challenges (homopolymers, etc)• Not diagnostic yet (Research use only)

• Low throughput• Labor-intensive• High cost (2835€ for NDM)

Multigene approach Sequential approach (gene by gene)