Figure 2

Mutational analysis. (A) 1^st hit detection: analysis of chromosome 22. (A.1) Chromosome 22 analysis by Multiplex Microsatellite PCR (MMP) using blood, T1 and T2 gDNA samples. Light blue line: heterozygous region; solid blue line: loss of heterozigosity (LOH); dashed line: LOH region not determined by MMP but confirmed by MLPA. Location of NF2, SMARCB1 and LZTR1 genes are indicated in green. Microsatellite position is indicated with a red line (o = no LOH, • = LOH). (A.2) Loss of chromosome 22 and LOH analysis characterized by SNP-array. A two-band pattern in the allele frequency plot indicates LOH from position 28570333 bp up to the telomere of chromosome 22q. Log2 Ratio < 0 indicates the loss of one copy of the same region. Breakpoint is indicated by a vertical grey dashed line. (B) Characterization of 2^nd hit NF2 mutations in both tumors. Mutations at DNA and RNA levels are shown. The effect of mutations on splicing alteration is schematically represented. Forward sequence of exon 3 at gDNA level from T1 (B.1) and exon 14 from T2 (B.2) are shown, accompanied by the effect at mRNA level and an schematic representation. (C) Haplotypes of patient’s family pedigree and analyzed tumors. MMP markers are indicated in blue. Chromosome 22q loss in tumors is indicated by a red box. Patient’s daughters inherited chromosome 22 carrying the first hit mutation in patient’s tumors but in its wild-type form.