Quantification of copy number of coding sequences of the LEPR gene was performed using a Salsa

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1 Supplemental Materials and Methods Characterization of the Δexon6-8 LEPR mutation MLPA analysis Quantification of copy number of coding sequences of the LEPR gene was performed using a Salsa MLPA P220 obesity kit (MRC Holland, Amsterdam, Netherlands) according to the manufacturer's instructions. MLPA PCR products were separated on an ABI 3730 automated sequencer (PE Applied Biosystems, Foster City, CA, USA). MLPA data were analyzed with GeneMapper 4.00 software. After population normalization, peak height for each LEPR gene exon analyzed with the kit was compared with a control sample. Peak heights below 0.6 were considered as heterozygous deletions and absence of peaks as homozygous deletions. qpcr analysis A dual color qpcr screening assay was set up on a LightCycler 480 Instrument (Roche Diagnosis, Mannheim, Germany). Dual Color Universal Probes for TBP (internal control, probe 2285, ENST , yellow 555 in 5 and dark quencher dye in 3, probe TBP kit) and for LEPR exon 6 (probe 22, FAM-labeled in 5 and dark quencher dye in 3 ) and Forward and Reverse primers for TBP and LEPR Exon 6 were used for quantitative amplification using an LC480 Probe Master Kit (Roche Diagnosis, Mannheim, Germany). Primer and probes for the Universal Probe Library assays were designed with Probe Finder software via the Universal Probe Library Assay Design Center at using the dual-color mode option. RT-PCR analysis The blood was collected in PET PAXgene tube (Becton Dickinson, Frankin Lakes, NJ, USA). This system is used to collect, store and stabilize the intracellular RNA of whole blood. The purification of intracellular RNA was performed using the PAXgene blood RNA kit (Qiagen, Venlo, Limburg, Netherlands). Total mrna extracted from leukocytes from c.2491g>a mutation carrier 4-II.1 and

2 from one control subject was reverse transcripted and the LEPR cdna was analyzed by PCR using forward primer in exon 16:(5 -GCAGTCACTCAGTGCTTATCC-3 ) and reverse primer in exon 19:(5 -TGAAAATTAAGTCCTTGTGCCC-3 ). Supplemental Results RT-PCR analysis of the c.2491g>a LEPR mutation The c.2491g>a was predicted to lead to a missense substitution (p.d831n) and to affect a splice donor site of exon 17-intron 18. We found one cdna product shorter than expected by RT-PCR analysis (data not shown). We sequenced this cdna and found the absence of exon 17 and the presence of exon 16 and 18 sequences, suggesting that the deletion would lead to a truncated protein without reading frameshift that should be noted p.h800_n831del. Nevertheless, we cannot exclude the co-existence of other spliced forms for this mutated mrna that would not have been amplified with the primers used. Supplemental Table 1: Baseline characteristics of the 535 unrelated obese French subjects. Age (mean in years ± SD) 22.6 ± 12.2 Sex 248 M, 287 F Nationality BMI (mean in kg/m 2 ± SD) 45.9 ± 12.2 F: female; M: male; SD: standard deviation French, 49 originated from the Reunion Island Supplemental Figure 1: Positions of the identified mutations in the LEPR protein. We observed five homozygous mutations in nine obese probands. Mutations marked by asterisks* were observed in compound heterozygous probands. The mutation indicated in red color is the Reunion Island mutation. Supplemental Figure 2: Mean BMI Z-score of homozygous, heterozygous and not mutated subjects for LEPR mutations in studied families.

3 BMI Z-scores were available for all homozygous subjects and for heterozygous and not mutated subjects from families 1, 3, 4, 6, 9 and 11. The BMI Z-scores were represented as the mean ± SEM (standard error of the mean). BMI Z-score is significantly higher in LEPR mutation homozygous carriers when compared to heterozygous and not mutated relatives (** p=0.0002).

4 Signal peptide P166CfsX7* Cytokine domain CRH1 domain Fibronectin III domain Immunoglobulin domain Extracellular domain Y422H* 535-1G>A* C604G N624KfsX21 T711NfsX18* L786P D831N Cytokine domain Fibronectin III domains CRH2 domain Cell membrane Intracellular domain Box 1 Box 2 Box 3 Supplemental Figure 1

5 SD BMI Z-score BMI Z-score SD Homozygotes n=12 Heterozygotes n=21 Not mutated n=11 Supplemental Figure 2