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. Itan, To evaluate Copy number variations (CNVs, i.e duplication and large deletion events, for each individual) the relative read count for each targeted region was determined as the ratio of the read count for that region divided by the total absolute read counts of all targeted regions of the design. The ratio of the relative read count of a region in a given individual over the average relative, Bioinformatics Core facility. After demultiplexing, sequences were aligned to the reference human genome hg19 using the Burrows-Wheeler Aligner. Downstream processing was carried out with the Genome Analysis Toolkit (GATK), SAMtools and Picard, following documented best practices, 2009.

, The mean depth of coverage per sample was >300X (except for patients 51, 148, 150, 152 and 155 for whom it was comprised between 267X and 298X) permitting to maximize the detection of variant in regions with less coverage and to enable accurate Copy Number Variant Analysis of the exons of the panel

, We excluded known variants with a minor allele frequency ?5% listed in databases or variants previously identified in "in-house" exomes. We subsequently selected for variants affecting splice sites or coding regions (nonsynonymous, nonsense, frameshift, start/stop gain or loss) and predicted to be damaging by in silico prediction tools. Additional prediction tools were then used to predict the pathogenicity of candidate missense variants identified (Panther, MutationAssessor, AlignGVGD, SNAP2, SNP&GO, Condel, FATHMM, Provean and MutPred2)

, Microsatellites genotyping Primers sequences of microsatellite markers (available at the Working Draft of the Human Genome available at UCSC, Human Assembly, p.19






, For experiments, keratinocytes were grown in 0.06 mM CaCl 2 EpiLife medium (Invitrogen) to 70-80% confluence. RNA isolation from primary human epidermal keratinocytes and RT-PCR Subconfluent cultured keratinocytes were harvested, RNA was prepared via RNeasy Mini (Qiagen), and cDNA was prepared with random hexamer priming and SuperScript IV reverse transcriptase (LifeTechnologies). PCR was performed in triplicate for, FAM-labelled amplified fragments were electrophoresed on an Applied Biosystems 3500XL genetic analyser using GeneScan? 400 HD ROX? Size Standard (Life technologies) and analyzed using the Gene-Mapper Software 5 (Applied Biosystems, 1987.

, Six control subjects were used, giving similar results. The experiment was replicated at least three times. PGK1 was used as an endogenous control

. Rt-pcr,

, Quantitative PCR was performed with PSENEN or HES1 and normalization to PGK1

R. F=ctgtggcttctggcatacct, Protein isolation from primary human epidermal keratinocytes and immunoblotting Keratinocytes were lysed in buffer (50 mM pH 8 Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 5 mM pH 8 EDTA pH8, Complete protease inhibitors [Roche]), incubated on ice for 30 min and sonicated for 2 min, p.1

T. Hcl and . Sds, 002% bromophenol blue), separated by SDS-PAGE, transferred onto Nitrocellulose membrane (Trans-Blot Turbo Nitrocellulose, BioRad), and incubated with primary and secondary antibodies, with chemiluminescence detection (ECL Plus Western Blotting Substrate, Pierce) using the ChemiDoc Imaging System (BioRad). ?-actin served as loading control. Experiments were replicated three times

, Antibodies: Primary antibodies included rabbit polyclonal anti-PEN2 (Enzo ADI-905-736), rabbit polyclonal anti-NCSTN (Sigma N1660) and mouse monoclonal anti-PSEN1

, Secondary antibodies were HRP-linked anti-rabbit or anti-mouse IgG antibodies (Cell Signaling #7074 and #7076)

, Statistical analyses: Statistical significance of observed differences was assessed by twotailed Mann-Whitney U tests, Means with s.e.m. are shown