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Supplementary Figure S1. NF1 copy number variations identified by the MLPA approach
were confirmed using a custom array-CGH. A. SALSA MLPA kits P081 NF1 peak areas
normalized ratio profiles of three patients. Two partial intragenic deletions and one partial
intragenic duplication were identified: NF00668 (deletion of exon 6 to exon 8), NF00735
(duplication of exon 28 to exon 29), and NF00587 (mosaic deletion of exon 1 to exon 35). B.
Array CGH profiles of the two partial deletions of patients NF00668 and NF00587 and the
partial duplication of patient NF00735. C. Zoom on breakpoints regions easily identified
rearrangements.
Supplementary Figure S2. Flow chart for NF1 comprehensive mutation screening. Prescreening of large NF1 deletions by four intragenic microsatellites genotyping leads to a
complete homozygosity situation in only 10% of the cases (step ). Around half of them
harbour a complete or partial deletion of the NF1 locus as confirmed by real-time PCR-based
gene dosage. Custom high resolution array-CGH enables the accurate characterization of the
deletion type. NF1 complete and large partial deletions were observed in 4.2% and 0.5% of
NF1 patients in the French cohort, respectively. In absence of large deletion, molecular
investigation of NF1 is achieved by a cDNA sequencing approach (step ). Point mutations
identified by this approach are observed in 88.2% of the NF1 patients. In front of a negative
screening of large deletion or point mutations which occurs in nearly 7% of the cases, a
quantitative analysis of nearly all the coding exons on genomic DNA is performed by multiple
ligation-dependent probe amplification (MLPA) analysis leading to the identification of single
or multi-exon deletions/duplications in 3.4% of the NF1 patients (step ). In case of a
negative result, the 57 constitutive coding exons and the alternatively spliced exon 31 are
sequenced on genomic DNA (step ). This comprehensive mutation screening enabled us to
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identify a NF1 mutation in 97% of the NF1 patients in the French cohort. This flow chart was
previously described in Sabbagh et al. 2013.14
Supplementary Figure S3. Patient NF00735 presented a direct tandem duplication of NF1
exons 28 to 29. NF1 exons named according to NCBI nomenclature (exons numbered 1-58)
are represented by rectangles (not proportional to their size) at the genomic level and at the
corresponding cDNA level. Exons 28 to 29 duplication was studied at the cDNA level. After
reverse transcription of NF1 mRNA, PCR was performed by using primers located in exon 21
(NF1_4U: 5’-AGTCCTGCTCTGTATCCAATG-3’) and in exon 34 (NF1_4L: 5’AGCACATTGCCGTCACTTAT-3’) for the upper and the lower primer, respectively. The
cDNA amplification was carried out on both wild type and mutated alleles and Sanger
sequencing confirmed the intragenic frameshift tandem repeat of NF1 exons 28 to 29 (266 bp)
in a direct orientation.
Supplementary Figure S4. Patient NF00966 presented a direct tandem duplication of NF1
exons 49 to 57. NF1 exons named according to NCBI nomenclature (exons numbered 1-58)
are represented by rectangles (not proportional to their size) at the genomic level and at the
corresponding cDNA level. Exons 49 to 57 duplication was studied at the cDNA level. After
reverse transcription of NF1 mRNA, PCR was performed by using primers located in exon 51
(NF1_51L: 5’-GGACCATGGCTGAGTCTCCTTT-3’) and in exon 55 (NF1_55U: 5’ACCCTGTTATCATTGTGCCAAGA-3’) for the lower and the upper primer, respectively.
The cDNA amplification could only be achieved in the presence of a tandem duplication in a
direct orientation. The specific PCR product overlapping the duplicated sequences junction
was obtained and Sanger sequencing confirmed the intragenic inframe tandem repeat of NF1
exons 49 to 57 (1188 bp).
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Supplementary Figure S5. NF1 amplicons (X axis) read depth normalized ratio (Y axis)
profiles of 22 NF1 patients DNA presenting NF1 intragenic deletion or duplication and four
normal control DNAs. Normalized ratios of < 0.6 and > 1.3 were considered as deletions and
duplications, respectively. Ratio profiles between 0.6 and 0.85 corresponded to a mosaic
deletion for patient NF00587.
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