460 Diabetes Care Volume 37, February 2014

Amelie´ Bonnefond,1,2,3,4 Julien Philippe,1,2,3 Highly Sensitive Diagnosis of 43 Emmanuelle Durand,1,2,3 Jean Muller,5,6 Sadia Saeed,7 Muhammad Arslan,8 Monogenic Forms of Diabetes or Rosa Mart´ınez,9 Franck De Graeve,1,2,3 Veronique´ Dhennin,1,2,3 Obesity Through One-Step PCR- Iandry Rabearivelo,1,2,3 Michel Polak,10,11 Hel´ ene` Cave,´ 12 Luis Castano,~ 9 Based Enrichment in Combination Martine Vaxillaire,1,2,3 Jean-Louis Mandel,5,6,13 Olivier Sand,1,2,3 With Next-Generation Sequencing and Philippe Froguel1,2,3,4,7

1European Genomic Institute for Diabetes, Lille, France 2CNRS UMR8199, Pasteur Institute of Lille, Lille, France 3Lille 2 University, Lille, France OBJECTIVE 4Qatar Biomedical Research Institute, Qatar Foundation, Doha, Qatar Accurate etiological diagnosis of monogenic forms of diabetes and obesity is 5Institut de Gen´ etique´ et de Biologie Moleculaire´ useful as it can lead to marked improvements in patient care and genetic coun- et Cellulaire, CNRS UMR7104, INSERM U964, seling. Currently, molecular diagnosis based on Sanger sequencing is restricted to Universite´ de Strasbourg, Illkirch, France 6 ˆ only a few genes, as this technology is expensive, time-consuming, and labor- Laboratoire de Diagnostic Gen´ etique,´ Hopitaux Universitaires de Strasbourg, Strasbourg, France intensive. High-throughput next-generation sequencing (NGS) provides an op- 7Department of Genomics of Common Disease, portunity to develop innovative cost-efficient methods for sensitive diabetes and Hammersmith Hospital, Imperial College obesity multigene screening. London, London, U.K. 8Department of Biological Sciences, Forman RESEARCH DESIGN AND METHODS Christian College, Lahore, Pakistan 9Cruces University Hospital, Universidad del Pa´ıs We assessed a new method based on PCR enrichment in microdroplets (Rain- Vasco/Euskal Herriko Unibertsitatea, Spanish Dance Technologies) and NGS using the Illumina HiSeq2000 for the molecular Biomedical Research Centre in Diabetes and EMERGING TECHNOLOGIES AND THERAPEUTICS diagnosis of 43 forms of monogenic diabetes or obesity. Forty patients carrying a Associated Metabolic Disorders, Barakaldo, known causal mutation for those subtypes according to diagnostic laboratories Spain 10INSERM U845, Universite´ Paris Descartes, were blindly reanalyzed. Sorbonne Paris Cite,´ Paris, France 11Department of Paediatric Endocrinology, RESULTS Necker Enfants-Malades Hospital, Assistance Except for one variant, we reidentified all causal mutations in each patient as- Publique-Hopitauxˆ de Paris, Paris, France 12Department of Genetics, Robert-Debre´ sociated with an almost-perfect sequencing of the targets (mean of 98.6%). We ˆ fi Hospital, Assistance Publique-Hopitaux de Paris, failed to call one highly complex indel, although we identi ed a dramatic drop of Paris, France coverage at this locus. In three patients, we detected other mutations with a 13Chaire de Gen´ etique´ Humaine, College` de putatively deleterious effect in addition to those reported by the genetic diag- France, Illkirch, France nostic laboratories. Corresponding author: Philippe Froguel, [email protected]. CONCLUSIONS Received 22 March 2013 and accepted 10 Our NGS approach provides an efficient means of highly sensitive screening for September 2013. mutations in genes associated with monogenic forms of diabetes and obesity. As This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/ cost and time to deliver results have been key barriers to uncovering a molecular suppl/doi:10.2337/dc13-0698/-/DC1. cause in the many undiagnosed cases likely to exist, the present methodology A.B. and J.P. contributed equally to this work. should be considered in patients displaying features of monogenic diabetes or © 2014 by the American Diabetes Association. obesity. See http://creativecommons.org/licenses/by- Diabetes Care 2014;37:460–467 | DOI: 10.2337/dc13-0698 nc-nd/3.0/ for details. care.diabetesjournals.org Bonnefond and Associates 461

Type 2 diabetes and obesity are complex cost-efficient, and rapid method to Primer Library Design disorders that are associated with identify causal mutations in patients We selected all susceptibility genes for several factors of genetic, epigenetic, with monogenic disorders (10). monogenic forms of diabetes or obesity, and environmental origins (1,2). Familial However, this technology is not which were known at the time of the aggregation of both type 2 diabetes and currently perfect for clinical molecular design (n = 43; Table 1 and obesity demonstrates a high heritability diagnosis, as it leads to marked gaps of Supplementary Table 1). All exons, (between 40 and 70%), which can make sequence (.5% of the target regions, including at least 40 base pairs (bp) of it difficult to select cases more likely to even with high mean depth of the flanking intron of each exon, and have a monogenic cause (1,3). However, sequencing coverage) (10,11), which is 1,000 bp upstream of exon 1 and deleterious coding gene mutations have problematic when the investigators are downstream of the last exon were been shown to cause almost totally looking for only one causal mutation. targeted. The primer library was penetrant severe forms of diabetes and In the current study, we aimed to designed using proprietary pipeline obesity, including neonatal diabetes assess a new method based on PCR developed by RainDance Technologies fl mellitus (NDM), maturity-onset enrichment in microfluidic droplets and (Lexington, MA). Brie y, primer diabetes of the young (MODY) and next-generation sequencing (NGS) for selection was based upon standard PCR several related syndromes like Bardet- the molecular diagnosis of 43 subtypes criteria, namely guanine-cytosine Biedl syndrome (BBS), Alstrom¨ of monogenic diabetes or obesity. content between 25 and 80%, amplicon syndrome (ALMS), Wolcott-Rallison, or A total of 40 patients carrying a known length between 200 and 600 bp, melting 8 Wolfram syndrome (4,5). All of these causal mutation for those subtypes temperature between 56 and 60 C, and monogenic forms of diabetes and according to genetic diagnostic primer length between 15 and 33 bp. obesity tend to occur at younger ages laboratories were blindly included in the Suitable primers were then screened and often exhibit other clinical features study. against the dbSNP131 database to (4,5). remove all primers that hybridized to polymorphism containing targeted In this context, an accurate molecular RESEARCH DESIGN AND METHODS sequences. The 542 targeted exons diagnosis of these extreme and often Patient Selection were covered by 970 primer pairs familial forms of diabetes and obesity is We selected a total of 40 patients (targeting a total of 336 kb of sequence) crucial for an optimal care of the presenting with a monogenic form of that were encapsulated into patients and genetic counseling for their diabetes (n = 19) or obesity (n = 21) who microfluidic droplets via RainDance families. The most striking example is were carriers of a known causal Technologies (Table 1). seen for the NDM patients carrying a mutation according to diagnostic heterozygous point mutation in the laboratories: 9 NDM patients, primarily Microdroplet-Based PCR Enrichment ABCC8 or KCNJ11 gene, encoding the assessed by the Department of Genetics The 40 DNA samples (3 mg) were two subunits (SUR1 and KIR6.2, in Robert-Debre´ Hospital (Assistance fragmented to 2–4 kb by sonication ˆ respectively) of the pancreatic b-cell– Publique-Hopitaux de Paris, Paris, (Bioruptor NGS; Diagenode, Liege,` + expressed ATP-dependent K channels. France); 10 MODY patients, primarily Belgium) and purified using the Indeed, these patients can be optimally assessed by the Cruces University MinElute system (Qiagen, Valencia, CA). treated by oral sulfonylurea drugs Hospital- Centro de Investigacion´ The quality of both fragmentation and instead of lifelong insulin therapy, Biomedica´ en Red de Diabetes y purification was assessed using the 2100 leading to remarkable improvements in Enfermedades Metabolicas´ Asociada Bioanalyzer (Agilent Technologies, glucose control and quality of life (6–8). (n = 5; Barakaldo, Spain) and the CNRS Santa Clara, CA). Subsequently, the Furthermore, a recent study UMR8199 unit (n = 5; Lille, France); 12 RainDance primer library was merged demonstrated that personalized genetic patients with an early-onset severe with each sheared genomic DNA sample medicine applied to patients with NDM, obesity, primarily assessed by the on the RDT1000 (RainDance which is currently based on standard Department of Genomics of Common Technologies), according to the Sanger sequencing of KCNJ11 and Disease in Imperial College of London manufacturer’s protocol. The resulting ABCC8 that is performed by clinical (n = 6; London, U.K.) and the CNRS emulsion, containing ;1million diagnostic laboratories (at a cost of UMR8199 unit (n = 6; Lille, France); and potential PCR reactions, was $2,815 in the U.S. [9]), leads to high 9 BBS patients, primarily assessed by the subsequently amplified by PCR on the financial benefits (9). However, as Genetic Diagnostic Laboratory in the Mastercycler Pro S (Eppendorf, ˆ KCNJ11 and ABCC8 encode a total of 40 Hopitaux Universitaires de Strasbourg Hamburg, Germany). The addition of coding exons, this genetic testing is (Strasbourg, France). Destabilizer reagent (RainDance obviously time-consuming, labor- The study protocol was approved by all Technologies) resulted in the intensive, and restricted to two genes local ethic committees, and study degradation of the emulsion and the only while NDM can be due to mutations participants signed an informed separation of oil and aqueous phases. in at least 11 genes (Table 1) (1,4). We consent. For children ,18 years, an oral A brief centrifugation completed this previously showed that whole-exome consent was obtained (when $3 years separation. PCR products were then sequencing (WES) was an attractive of age), and the parents provided purified using MinElute columns alternative as it was a comprehensive, written informed consent. (Qiagen) and assessed for both quality 462 Diagnosis of Monogenic Diabetes and Obesity Diabetes Care Volume 37, February 2014

Table 1—List of the targeted susceptibility genes for monogenic forms of diabetes or obesity and median read depth of the targeted regions Number of RDT Target RDT design Median read Gene Disease Inheritance* Gene location amplicons size (bp) size (bp) depth (IQR)† ABCC8 NDM/MODY12 (S or AD)/AD 11p15.1 43 8,711 13,325 1,007 (669–1,353) CEL MODY8 AD 9q34.3 20 4,934 8,035 831 (473–1,173) EIF2AK3 WRS AR 2p12 25 7,498 11,501 271 (137–454) FOXP3 IPEXS XR Xp11.23 18 4,827 7,035 930 (580–1,366) GCK MODY2/NDM AD/AR 7p15.3-p15.1 21 6,673 9,261 843 (584–1,267) GLIS3 sNDM AR 9p24.2 32 11,367 16,431 555 (349–804) HNF1A MODY3 AD 12q24.2 16 5,738 7,011 866 (560–1,204) HNF1B MODY5/sNDM AD/(S or AD) 17q12 15 5,286 6,776 696 (474–1,009) HNF4A MODY1 AD 20q13.12 23 8,224 10,858 573 (414–1,250) INS NDM/MODY10 (S, AD or AR)/AD 11p15.5 8 2,744 4,136 405 (340–496) KCNJ11 NDM/MODY13 (S or AD)/AD 11p15.1 13 6,133 7,549 494 (395–577) NEUROD1 MODY6/sNDM AD/AR 2q32 11 5,045 6,127 324 (237–529) NEUROG3 sNDM AR 10q21.3 8 3,355 4,533 415 (330–491) PDX1 NDM/MODY4 AR/AD 13q12.1 11 4,673 6,341 338 (285–380) PTF1A sNDM AR 10p12.2 8 3,433 4,647 350 (285–418) RFX6 sNDM AR 6q22.1 26 6,329 10,506 390 (188–559) WFS1 WS AR 4p16.1 18 6,040 8,592 846 (604–1,305) ALMS1 ALMS AR 2p13 45 16,072 21,600 331 (229–491) ARL6 BBS3 AR 3q11.2 15 3,953 6,199 244 (150–371) BBS1 BBS1 AR 11q13 24 6,123 9,029 886 (544–1,259) BBS2 BBS2 AR 16q21 22 5,664 8,208 575 (337–859) BBS4 BBS4 AR 15q22.3-q23 22 5,292 7,957 639 (428–811) BBS5 BBS5 AR 2q31.1 22 5,583 9,194 221 (137–380) BBS7 BBS7 AR 4q27 29 8,232 12,644 220 (125–344) BBS9 BBS9 AR 7p14 29 7,169 11,157 265 (196–443) BBS10 BBS10 AR 12q21.2 12 5,683 7,020 278 (180–347) BBS12 BBS12 AR 4q27 13 5,582 7,200 244 (198–288) BDNF SEO S 11p13 31 14,038 17,550 300 (237–364) CEP290 BBS14 AR 12q21.32 59 12,517 24,326 139 (57–282) GNAS AHO AD (from the mother) 20q13.3 35 11,439 16,089 807 (412–1,192) LEP SEO AR 7q31.3 13 5,577 7,119 462 (355–625) LEPR SEO AR 1p31 41 13,803 19,329 244 (138–371) MC4R SEO AD or AR 18q22 7 3,488 4,124 253 (221–308) MKKS BBS6 AR 20p12 16 6,123 8,298 309 (244–435) MKS1 BBS13 AR 17q22 23 5,440 7,937 980 (665–1,264) NTRK2 SEO S 9q22.1 59 22,705 29,033 520 (366–758) PCSK1 SEO AD or AR 5q15-q21 29 9,761 13,253 438 (284–685) POMC SEO AR 2p23.3 9 3,495 4,595 621 (407–1,112) SDCCAG8 BBS16 AR 1q43 22 5,517 8,347 389 (198–650) SIM1 SEO/PWL AD 6q16.3-q21 19 6,545 8,382 672 (414–1,069) TRIM32 BBS11 AR 9q33.1 13 5,819 7,344 393 (316–488) TTC8 BBS8 AR 14q31.3 20 4,953 8,029 293 (175–442) WDPCP BBS15 AR 2p15 25 6,209 10,319 244 (99–451) Total 970 307,792 436,946 427 (231–781) AHO, Albright hereditary osteodystrophy; IPEXS, immune dysregulation polyendrocrinopathy enteropathy X-linked syndrome; IQR, interquartile range; PWL, Prader-Willi-like syndrome; SEO, severe early-onset obesity; sNDM, syndromic NDM; RDT, RainDance technology; WRS, Wolcott- Rallison syndrome; WS, Wolfram syndrome. *Mode of inheritance: AD, autosomal dominant; AR, autosomal recessive; S, spontaneous; XR, X-linked recessive. †Median read depth of the target regions per gene (in the 40 samples). care.diabetesjournals.org Bonnefond and Associates 463

and predicted amplification profile on mapped to the reference human per gene was 4273 (Table 1). In each the 2100 Bioanalyzer (Agilent genome (UCSC NCBI37/hg19) using the DNA sample, 98.6 6 0.8% of the Technologies). pipeline CASAVA (version 1.8.2). Variant targeted regions were successfully detection was performed with the same sequenced with a depth of coverage Targeted Capture for WES pipeline and filtered to reach depth of at $83 (Table 2). We successfully m Two DNA samples (3 g) were also least 83. In both samples, we sequenced 96.1 6 1.9% and 95.1 6 captured using the Agilent SureSelect obtained a mean depth of coverage of at 2.6% of the targeted regions when we Human All Exon Kit (version v4; Agilent least 2003. fixed more stringent depths of coverage Technologies), following the All detected variants were annotated $203 and $303, respectively. When manufacturer’s protocols and as using the Ensembl Perl API (version 66). we considered coding exons only, 508 previously described (11). exons out of a total of 520 were Variant Prioritization perfectly sequenced for all participants Illumina Library Preparation and For each participant, after the detection (Fig. 1), while nine exons were partially Sequencing of all variants in the targeted regions, we PCR products were repaired to blunt sequenced (in CEL, BBS9, CEP290/BBS14 focused on rare variants of interest (i.e., ends and concatenated using the NEB LEPR,andTTC8/BBS8; Fig. 1) and three nonsynonymous variants, essential Quick blunting kit and the NEB Quick exons were poorly sequenced in all splice-site variants, or indels leading to a ligation kit (New England Biolabs, participants (in BBS7 and CEP290/ gain of STOP codon and nonsense Ipswich, MA). The samples were then BBS14; Fig. 1). Therefore, the variants) with minor allele frequency purified using Agencourt AMPure XP sequencing problems using the present ,1% (or “not available”) according to beads (Beckman Coulter, Fullerton, CA) technology were not random but the Single Nucleotide Polymorphism fi and fragmented to ;200 bp by seemed to be speci c to few exons. Of database #135 (dbSNP135), the 1000 sonication (Bioruptor NGS; Diagenode). note, the failed sequence regions were Genomes project (12), and the National The quality of fragmentation and not rich in guanine and cytosine Heart, Lung, and Blood Institute Grand purification was assessed using the 2100 (between 20 and 40% of guanine- Opportunity Exome Sequencing Project Bioanalyzer (Agilent Technologies). cytosine). Two DNA samples were (ESP) (13). We primarily looked for Adaptors were ligated to the fragments sequenced through RainDance variants of interest located in genes by using NEBNext Multiplex Oligos for Technologies in combination with related to the main phenotype of the Illumina (New England Biolabs). Finally, Illumina sequencing and through patient (Table 1). Furthermore, we the 40-amplicon enriched samples were Agilent SureSelect All Exon kit (Agilent checked if there were additional sequenced on the HiSeq2000 (Illumina, Technologies) in combination with variants of interest in the other genes of San Diego, CA) in 76-bp paired-end Illumina sequencing. In both samples, the panel, according to the model of reads by multiplexing 12 samples per 100% of coding variants that were inheritance leading to the disease lane, while the two whole-exome– identified by WES were also found using (presence of homozygous mutations in enriched samples were sequenced on RainDance Technologies in combination recessive models or presence of the HiSeq2500 (Illumina) in 100-bp with Illumina sequencing. heterozygous mutations in dominant paired-end reads by multiplexing the models; Table 1). As a blind test, the investigators only two samples in two lanes. knew the main phenotypes of the 40 Sanger Sequencing patients but not the known causal Data Analysis When we found additional variants mutation carried by these patients Demultiplexing of sequence data were with a putatively deleterious effect, we (according to the different genetic performed with CASAVA (version 1.8.2). confirmed them by Sanger sequencing diagnostic laboratories). Except for a Regarding the 40-amplicon enriched on a 3730xl DNA Analyzer (Applied patient presenting with BBS (patient samples, sequence reads were mapped Biosystems, Foster City, CA). A standard 21), we reidentified the putatively to the human genome (UCSC NCBI37/ protocol was performed. Primer designs causal mutation in every participant, hg19) using the Burrows-Wheeler and PCR conditions can be provided which had primarily been detected Aligner (version 0.6.1; algorithm "BWA- upon request. Sequencing reads were by genetic diagnostic laboratories SW"; default parameters) to generate a assembled and analyzed using Variant (Table 2). The list of these causal BAM file. This aligner deals with Reporter software (Applied mutations included: 23 nonsynonymous chimeric reads generated by the library Biosystems). variants, 7 nonsense variants, preparation method described above. 4 splice-site variants, and 10 indels Variant calling was performed with RESULTS (involving 1–5 nucleotides) (Table 2). SAMtools (version 0.1.18; algorithm In genomic DNA samples from 40 Our algorithm failed to detect a "pileup" with the following parameters: patients presenting with an elucidated highly complex homozygous mutation -u -g -B -m 3 -C 50 -d 1000000 -L 1000000 monogenic form of diabetes or obesity, in patient 21, BBS5-c.572_594inv -F 0.0002 -Q 0), when depth of we sequenced a total of 43 genes (Table {ins567_568};595_603del / $ 3 sequencing coverage was 8 . 1) using PCR enrichment in microfluidic p.His180Tyrfs*2, which was previously Regarding the two whole-exome– droplets followed by NGS. The overall described (14). However, when we enriched samples, sequence reads were median depth of sequencing coverage investigated the BAM file for this 464 Diagnosis of Monogenic Diabetes and Obesity Diabetes Care Volume 37, February 2014

Table 2—Sequence quality and list of the putatively causal mutations that have been detected per participant Percent targeted Putatively causal mutation(s) Patient regions with Number of detected Known number Disease coverage $83 variants of interest* Mutation Status Gene† mutation‡ 1 NDM 99.3 50 c.683G.C p.Gly228Ala het KCNJ11 U c.674T.C p.Leu225Pro het ABCC8 2 NDM 99.4 38 c.545T.C p.Ile182Thr het KCNJ11 U 3 NDM 99.1 51 c.3067C.T p.His1023Tyr het ABCC8 U 4 NDM 98.8 46 c.2473C.T p.Arg825Trp het ABCC8 U 5 NDM 99.1 43 c.638T.G p.Leu213Arg het ABCC8 U 6 NDM 99.0 49 c.3544C.T p.Arg1182Trp het ABCC8 U 7 NDM 98.9 46 c.4567G.A p.Val1523Met het ABCC8 U 8 NDM 99.3 41 c.287G.A p.Cys96Tyr het INS U 9 NDM 99.3 49 c.265C.T p.Arg89Cys het INS U 10 MODY 99.4 59 c.1144C.T p.Gln382* het HNF1B U 11 MODY 98.5 45 c.207_211del p.His69Glnfs*17 het HNF1B U 12 MODY 99.3 48 c.679+1G.A d het GCK U 13 MODY 98.7 48 c.92G.A p.Gly31Asp het HNF1A U 14 MODY 98.7 51 c.685C.T p.Arg229* het HNF1A U 15 MODY 97.7 46 c.835G.T p.Glu279* het GCK U 16 MODY 97.8 57 c.437T.G p.Leu146Arg het GCK U 17 MODY 99.1 49 c.476G.A p.Arg159Gln het HNF1A U 18 MODY 97.9 57 c.19C.T p.Gln7* het HNF1A U 19 MODY 97.8 48 c.1079C.A p.Ser360* het GCK U 20 BBS 98.9 41 c.792T.G p.Tyr264* hom MKKS U 21 BBS 98.6 43 ? ? ? ? 22 BBS 98.6 51 c.3G.A p.Met1Ile het MKKS U c.110A.G p.Tyr37Cys het MKKS U c.2083A.G p.Thr695Ala het ABCC8 c.182T.G p.Val61Gly het HNF1B 23 BBS 99.4 49 c.271dup p.Cys91Leufs*5 het BBS10 U c.728_731del p.Lys243Ilefs*15 het BBS10 U 24 BBS 98.1 53 c.221–2A.T d hom BBS4 U 25 BBS 98.7 53 c.1044_1045del p.Pro350Ilefs*11 het BBS10 U c.2065A.C p.Thr689Pro het BBS10 U 26 BBS 99.5 56 c.113–2A.G d het BBS9 U c.263+4A.C d het BBS9 U 27 BBS 99.3 40 c.271dup p.Cys91Leufs*5 het BBS10 U c.1012G.T p.Glu338* het BBS10 U 28 BBS 99.5 51 c.1169T.G p.Met390Arg hom BBS1 U 29 SEO 97.0 44 c.482T.C p.Met161Thr hom MC4R U c.2264G.A p.Arg755Gln het ABCC8 30 SEO 98.9 46 c.104_106del p.Ile35del het LEP U 31 SEO 97.0 52 c.104_106del p.Ile35del het LEP U 32 SEO 96.8 48 c.104_106del p.Ile35del hom LEP U 33 SEO 96.9 44 c.398del p.Gly133Valfs*15 hom LEP U 34 SEO 97.0 48 c.398del p.Gly133Valfs*15 hom LEP U 35 SEO 98.3 43 c.1672A.G p.Thr558Ala het PCSK1 U 36 SEO 97.9 45 c.524C.T p.Thr175Met het PCSK1 U 37 SEO 99.0 42 c.967C.T p.His323Tyr het SIM1 U 38 SEO 98.4 46 c.974G.A p.Ser325Asn het PCSK1 U 39 SEO 98.9 50 c.731C.A p.Ala244Glu het MC4R U 40 SEO 98.6 55 c.2140A.G p.Thr714Ala het SIM1 U het, heterozygous; hom, homozygous; SEO, severe early-onset obesity. *Variants of interest include nonsynonymous variants, splice-site variants, indels, and nonsense variants. †Transcript identifications (NM) are reported in Supplementary Table 1. ‡Mutation that was primarily detected by the diagnostic laboratory in the patient. care.diabetesjournals.org Bonnefond and Associates 465

Figure 1—Sequence quality in targeted coding exons per gene in all patients. , 100% of coding exons were accurately sequenced; , between 10 and 99% of coding exons were accurately sequenced; and , less than 10% of coding exons were accurately sequenced. patient, we found a dramatic gap of associated with monogenic diabetes: CONCLUSIONS coverage at the locus of the mutation ABCC8-p.Thr695Ala and HNF1B-p. Currently, the methods of molecular (Supplementary Fig. 1). Val61Gly (Table 2). The ABCC8-p. diagnosis based on Sanger sequencing Importantly, in three individuals, we Thr695Ala mutation was novel and are restricted (when performed) to surprisingly detected other mutations predicted to be damaging according to only a few loci as this technology is quite with a putatively deleterious effect, in both LRT and MutationTaster software expensive, long, and tedious. Since an addition to those previously reported by (Table 3). The HNF1B-p.Val61Gly (or accurate etiological diagnosis of severe the genetic diagnostic laboratories. rs147816724) showed a minor allele forms of diabetes and obesity has been Indeed, in patient 1 presenting with frequency of 0.07% in European- proven to lead to marked improvements NDM (and diagnosed with a KCNJ11 American participants from the ESP in patient care and in family counseling, mutation), we also found a missense project (Table 3). Previous studies the development of cost-efficient, fast, mutation in ABCC8 (p.Leu225Pro), also reported that this mutation led to and high-throughput methods for an known as rs1048095 (Tables 2 and 3). kidney disease and uterine accurate DNA sequencing is of major According to PolyPhen-2 (15), SIFT (16), abnormalities (21,22). In patient 29 medical interest in metabolic diseases LRT (17) and MutationTaster (18) presenting with severe early-onset and beyond. In the current study, we software, the ABCC8-p.Leu225Pro was obesity and diagnosed with an MC4R assessed the PCR-based technology in fl predicted to be damaging (Table 3), and homozygous mutation, we detected a micro uidic droplets in combination several studies reported this mutation novel missense heterozygous mutation with NGS so as to achieve these goals. in patients presenting with NDM in ABCC8 (p.Arg755Gln), which had not We found this method very accurate as (6,19,20). Furthermore, Masia et al. (19) been detected by the 1000 Genomes or we were able to reidentify almost all showed that the mutation contributed ESP project and was predicted to be known mutations in each sample (44 of to overactivity of the ATP-dependent K+ damaging according to the LRT software 45). Furthermore, most of the targeted channel. In patient 22 diagnosed with a (Tables 2 and 3). Unfortunately, we did coding regions were perfectly BBS6 mutation, two additional missense not have access to additional clinical sequenced in all participants (Fig. 1). To mutations were detected in genes features for patients 1, 22, and 29. achieve even better results in gene 466 Diagnosis of Monogenic Diabetes and Obesity Diabetes Care Volume 37, February 2014

screening, the design of the current methodology will be able to improve panel of primers can easily be this poor diagnosis. reoptimized, designing new primer pairs Moreover, the present method is very for failed (or partly failed) amplicons. fast: for one sample, the microdroplet- Furthermore, when a small set of based PCR enrichment, the sequencing, regions fails (as in our study), it is quite and the analysis can be performed in 2 fi feasible to ll in these rarely occurring weeks only. gaps using a standard protocol of Sanger fi sequencing. A nearly perfect diagnosis Finally, we were able to nd more than protocol can therefore be defined for one variant with a putatively deleterious monogenic diabetes and obesity genes. effect in three patients, which should Of note, we only failed to call a highly help us to understand better the range complex indel in one BBS patient, of phenotypes found in families with fi early onset forms of diabetes or obesity. not available; PPh2, PolyPhen-2 software; SEO, although we identi ed a dramatic drop of coverage at the indel locus Indeed, given current sequencing (Supplementary Figure 1). While waiting progress, it becomes clearer that the for improvements in NGS algorithms of phenotypes associated with so-called indel calling and detection (that should monogenic diseases can be due to a set come soon), it is necessary to double- of penetrant mutations in several check the uniformity of coverage causative genes. In a recent study, throughout the sequence targets in through RainDance PCR-based negative samples, and it would be enrichment in combination with NGS, helpful to perform a standard multiplex Schrauwen et al. (24) also identified two ligation-dependent probe amplification causal mutations in two different genes to surely dismiss any presence of large in a patient presenting with hearing loss. indels. We believe that the assessment of all known susceptibility genes for a disease The cost of this method is very in only one step will bring a less-biased reasonable compared with Sanger analysis of the causes of the disease and sequencing or WES. When considering an obvious benefit for the patient (and reagents only and one DNA sample, the possibly his/her family if several RainDance PCR-based enrichment in putatively deleterious mutations are combination with NGS was five times present in the same pedigree). less expensive than WES ($330 vs. 1,630; based on the sequencing of 12 The present panel could primarily be RainDance amplicon-enriched libraries tested in patients presenting with: per lane and two whole-exome– 1) NDM (with an onset of diabetes ,1year enriched libraries per lane, respectively, of age); 2) a potential MODY (according using the Illumina HiSeq2000) and 20 to known guidelines and algorithms); times less expensive than Sanger 3) a potential BBS or ALMS; 4) severe sequencing with forward and reverse early-onset or familial obesity (in at least (NM_000458.2) NA 0.07% 0 0 1 1 Yes, in kidney disease and uterine abnormalities (21,22) (NM_000352.3) NA NA 1 1 1 1 Yes, in NDM (6,19,20) (NM_000352.3) NA NA 0 0 1 1 No (NM_000352.3) NA NA 0 0 1 0 No sequence for 970 amplicons ($330 vs. three generations); or 5) severe early- 6,783). Of note, the RainDance panel is onset obesity in a family with no other ABCC8 ABCC8 ABCC8 HNF1B conceived for 1,500 samples. Therefore, obesity case in siblings and parents (to if the number of samples is restricted, find a putative de novo mutation). After then the experiment is less cost- detection of all variants, the clinical efficient. However, the library can geneticist could pick up the potential A/p.Arg755Gln G/p.Thr695Ala include 20,000 amplicons (the present causal rare variants by: 1) focusing on C/p.Leu225Pro G/p.Val61Gly . . . . library includes 970 amplicons only). variants previously described in the Thus, additional genes involved in other literature or on novel variants not found

c.182T rare disorders could be easily added to in dbSNP, the 1000 Genomes project, or design a master panel applicable to ESP project; 2) assessing extended several dozens of rare diseases. family members to verify inheritance Furthermore, it is noteworthy that the pattern and/or segregation of the number of patients presenting with mutation with the phenotype; 3)using Characteristics of the additional mutations of interest

— monogenic diabetes or obesity is totally in silico damage prediction and underestimated. Shields et al. (23) conservation analyses; and, if possible, reported that .80% of MODY cases 4) performing functional studies in cell Table 3 Patient number Disease1 Mutation NDM c.674T Gene MAF 1000G MAF ESP PPh2* SIFT* LRT* MutationTaster* Previous related studies 22 BBS c.2083A severe early-onset obesity. *Predicted to be: 0, not damaging; or 1, damaging. 29 SEO c.2267G 1000G, 1000 Genomes project; MAF, minor allele frequency in the European Utah residents with Northern and Western European ancestry population; NA, were undiagnosed. The present lines or animal models. care.diabetesjournals.org Bonnefond and Associates 467

A limitation of our assay may be the use manuscript, and managed the primary genetic 13. Tennessen JA, Bigham AW, O’Connor TD, of a coverage threshold of 83 for analyses on the participants of the current et al.; Broad GO; Seattle GO; NHLBI Exome fi variant detection. A recent study has study. All authors read and approved the nal Sequencing Project. Evolution and version of the present draft. P.F. is the functional impact of rare coding variation reported that raising the coverage guarantor of this work and, as such, had full from deep sequencing of human exomes. threshold to 133 markedly limited the access to all the data in the study and takes Science 2012;337:64–69 responsibility for the integrity of the data and number of missing variant calls (25). 14. Redin C, Le Gras S, Mhamdi O, et al. the accuracy of the data analysis. 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N Engl J Mutations in the hepatocyte nuclear factor- 10-LABX-46 and ANR-10-EQPX-07-01), the Med 2006;355:467–477 1b (HNF1B) gene are common with European EurOCHIP obesity FP7 Consortium, 9. Greeley SAW, John PM, Winn AN, et al. The combined uterine and renal malformations and the transnational European research grant cost-effectiveness of personalized genetic but are not found with isolated uterine on Rare Diseases (ERANET-09-RARE-005). medicine: the case of genetic testing in malformations. Am J Obstet Gynecol 2010; Duality of Interest. No potential conflicts of neonatal diabetes. Diabetes Care 2011;34: 203:364.e1–5 interest relevant to this article were reported. 622–627 23. Shields BM, Hicks S, Shepherd MH, Author Contributions. A.B. designed the 10. Bonnefond A, Durand E, Sand O, et al. Colclough K, Hattersley AT, Ellard S. study and wrote the manuscript. J.P. performed Molecular diagnosis of neonatal diabetes Maturity-onset diabetes of the young the PCR-based enrichment and NGS and wrote mellitus using next-generation sequencing (MODY): how many cases are we missing? the manuscript. E.D. performed the PCR-based of the whole exome. PLoS ONE 2010;5: Diabetologia 2010;53:2504–2508 enrichment and NGS. J.M., M.A., R.M., H.C., L.C., e13630 24. Schrauwen I, Sommen M, Corneveaux JJ, and M.V. managed the primary genetic analyses 11. Bonnefond A, Philippe J, Durand E, et al. et al. A sensitive and specific diagnostic test on the participants of the current study, Whole-exome sequencing and high for hearing loss using a microdroplet PCR- reviewed the manuscript, and contributed to throughput genotyping identified KCNJ11 based approach and next generation discussion. S.S., M.P., and J.-L.M. managed the as the thirteenth MODY gene. PLoS ONE sequencing. Am J Med Genet A 2013;161A: primary genetic analyses on the participants of 2012;7:e37423 145–152 the current study. F.D.G. and I.R. performed the bioinformatics analyses. V.D. reviewed the 12. Abecasis GR, Auton A, Brooks LD, et al.; 25. Meynert AM, Bicknell LS, Hurles ME, manuscript and contributed to discussion. O.S. 1000 Genomes Project Consortium. An Jackson AP, Taylor MS. Quantifying single performed the bioinformatics analyses, integrated map of genetic variation from nucleotide variant detection sensitivity in reviewed the manuscript, and contributed to 1,092 human genomes. Nature 2012;491: exome sequencing. BMC Bioinformatics discussion. P.F. designed the study, wrote the 56–65 2013;14:195