Venn diagram displaying the total number of variants in the targeted genes (coding exons and UTRs, with 10 bp of intronic flanking regions), which were identified by each enrichment method, in Patient #1 and Patient #2.

<p>The presence of mutations colored in red was not confirmed by Sanger sequencing while the presence of mutations colored in green was confirmed by Sanger sequencing (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143373#pone.0143373.s001" target="_blank">S1 Fig</a></b>).</p

Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in UTRs of targeted genes, according to each enrichment method.

<p>Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in UTRs of targeted genes, according to each enrichment method.</p

Number of UTRs (<i>n</i>_<i>total</i> = 197) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.

<p>Number of UTRs (<i>n</i>_<i>total</i> = 197) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.</p

Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in coding regions of targeted genes (with 10 bp of intronic flanking regions), according to each enrichment method.

<p>Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in coding regions of targeted genes (with 10 bp of intronic flanking regions), according to each enrichment method.</p

Number of coding exons (with 10 bp of intronic flanking regions; <i>n</i>_<i>total</i> = 576) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.

<p>Number of coding exons (with 10 bp of intronic flanking regions; <i>n</i>_<i>total</i> = 576) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.</p

Genotype correlation of <i>NR2F2</i> rs3743462 polymorphism on glucose homeostasis parameters and height in the French prospective DESIR cohort using up to four repeated measurements over the 9-year follow-up study.

*<p>The <i>P</i> values and β-coefficients are from the “mixed” regression model of each trait against genotype with age, gender and BMI as covariates (except for height adjusted for age and gender). The <i>P</i>-values indicated are nominal <i>P</i>-values.</p><p>The β-coefficient denotes the effect of rs3743462 minor C-allele and genotypes (depending on the genetic model tested) on the traits analyzed, i.e. the increase or decrease of the mean value for a specific trait.</p

NR2F2 binds the variant rs3743462-C oligonucleotide with higher affinity than the rs3743462-T oligonucleotide and the C-allele is associated with a strong decrease of <i>NR2F2</i> gene expression relative to the T-allele.

<p>(A) Multiple alignments of the genomic region between nucleotides −3180 and −3110 of the <i>NR2F2</i> gene regulatory regions present in the −3210/+873 construct. Deletion is indicated by dashes and points indicate identities. The sequence of the human complementary strand is shown above other sequences. Genomic sequences can be retrieved from GenBank by their accession codes: <i>Homo sapiens</i> (NT_010274.17|:11836273-11840385), <i>Mus musculus</i> (NT_039428.7|Mm7_39468_37:c10507606-10503510; reverse/complementary strand), <i>Rattus norvegicus</i> (NW_047560.2|Rn1_WGA2082_4:c5641306-5636690; reverse/complementary strand). The position of the human SNP is indicated by an asteriskabove the sequences of each species: <i>H. sapiens</i>, −3,138; <i>M. musculus</i>, −3,139; <i>R. norvegicus</i>, −3,152 (where transcription start site is +1). (B) The sense strand sequences (+) of the oligonucleotides used in EMSA are shown. SNP base pairs are shown in lower case letters. (C) The labeled rs3743462-T and rs3743462-C oligonucleotides were incubated with INS-1 832/13 nuclear extracts, and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (D) Comparison of the affinity of protein binding to the rs3743462-T and rs3743462-C variants. The labeled rs3743462-C oligonucleotide (Fig. 2B) was incubated with or without the indicated molar excess of unlabeled rs3743462-T or rs3743462-C oligonucleotide as competitors before addition of INS-1 832/13 nuclear extract. Protein binding was then analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (E) INS-1 832/13 nuclear extracts were incubated with or without of the indicated anti-serum. The labeled oligonucleotide representing the −3153/−3126 <i>NR2F2</i> regulatory region and containing the rs3743462-C allele was added and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (F) Functional analysis of the rs3743462 alleles in pancreatic β-cells. INS-1 832/13 cells were transiently co-transfected using lipofectamine solution containing either rs3743462 T-allele, C-allele, T-allele with DR-1 mutated site or C-allele with DR-1 mutated site within the context of the 3210/+873 sequences (1.5 µg) and expression vector encoding <i>Renilla</i> luciferase (0.1mg). Cells were then cultured in the presence of 5 mM glucose for 14 h. Results are calculated from the ratio of luciferase/<i>Renilla</i> activity. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences in expression at P<0.05.</p

Glucose decreases the ability of HNF4α to activate the transcription of the <i>NR2F2</i> proximal promoter and to bind its chromatin target.

<p>(A) INS-1 832/13 cells were transiently co-transfected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035810#pone.0035810-Perilhou2" target="_blank">[7]</a> with a luciferase reporter gene driven by various lengths of the <i>NR2F2</i> promoter, designated by their 5′ and 3′ end positions relative to the defined <i>NR2F2</i> gene transcription initiation site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035810#pone.0035810-Soosaar1" target="_blank">[25]</a> and a control vector expressing <i>Renilla</i> luciferase. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Results were calculated from the ratio of luciferase/<i>Renilla</i> activity. Background expression was subtracted using the mean expression level of empty pGL3-basic. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences, P<0.05. (B) Comparison of mRNA levels of NR2F2 and HNF4α relative to those of cyclophilin determined by real-time RT-QPCR in INS-1 832/13 β-cells stimulated with 5 mM (white bars) or 20 mM glucose (black bars) for 24 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035810#pone.0035810-Perilhou1" target="_blank">[3]</a> (C) ChIP from INS-1 832/13 cells cultured in the presence of either 5 mM or 20 mM glucose. Targets for QPCR amplifications were the proximal <i>NR2F2</i> promoter containing the DR1 DNA binding site or a downstream coding region (negative control). Amount of target chromatin precipitated by the HNF4α-specific antibody relative to that precipitated by control IgG (mean of three independent experiments ± SEM). *Significant difference, P<0.05. (D) COS cells were transiently co-transfected with the −328/+873 construct and 50 ng of empty KS vector (white bars), or HNF4α (grey bars) expression vectors. Means ± SEM of results from at least three separate transfections performed in triplicate are shown. *Significant difference, P<0.05. (E) INS-1 832/13 cells were transiently co-transfected with the wild-type or mutated −328/+873 constructs containing the DR1 elements. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Means ± SEM of results from ten independent transfections performed in triplicate are shown. *Statistically significant differences in percentage of expression (where value for 5 mM glucose is 100% relative to −328/+873 construct) at P<0.05.</p

Number of variants identified through the WES analysis of the four DNA samples.

a<p>This includes all identified variants (including insertion or deletion) that reach the quality threshold and with depth of coverage ≥8×; <b><i>Novel</i></b> means not present in the public database dbSNP130; <b><i>Indel</i></b>, insertion or deletion.</p

Pedigree of the family showing diabetes status of each member, as well as genetic status, age of diagnosis, treatment and date of birth.

<p>With regard to the genetic status, NM denotes the presence of the heterozygous <i>KCNJ11</i> p.Glu227Lys mutation and NN denotes the absence of mutation at the same locus. Circles represent female participants and squares male participants. A slash through the symbol indicates that the family member is deceased. Black symbols indicate patients with non autoimmune diabetes. The half-filled and quarter-filled symbols indicate individuals with impaired glucose tolerance and impaired fasting glucose, respectively. The black symbols with a white diagonal denote patients with type 1 diabetes. Green arrows point to members for whom the whole-exome was sequenced. <b><i>INS</i></b>, insulin; <b><i>OHA</i></b>, oral hypoglycaemic agents; <b><i>SU</i></b>, sulfonylurea.</p