Germline Epigenetic Silencing of the Tumor Suppressor Gene PTPRJ in Early-Onset Familial Colorectal Cancer  Ramprasath Venkatachalam  Gastroenterology  Volume 139, Issue 6, Pages 2221-2224 (December 2010) DOI: 10.1053/j.gastro.2010.08.063 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 An inherited duplication affecting the protein tyrosine phosphatase gene PTPRJ. (A) Pedigree showing the index patient (III:4, arrow) and her grandmother (I:2), both with colorectal cancer at 36 and 65 years of age, respectively (closed symbols). Deceased persons are indicated by a diagonal line. (B) Copy number profile of a segment of chromosome 11 and structural organization of the PTPRJ gene, showing the position of the duplicated region affecting the 5′ part of the gene. (C) Validation and segregation analyses by MLPA showing that the constitutional duplication in the index patient (green line) is also present in its tumor DNA (blue line) and the germline DNA of the unaffected father (II:1; purple line; currently 71 years old). Germline DNA from the mother (II:2 pink line) does not show the duplication. Black lines represent normal control DNAs. MLPA probes (available upon request) were designed according to guidelines provided by MRC-Holland (Amsterdam, The Netherlands). (D) Sequence analysis of the cloned 180-bp PCR fragment revealing the exact breakpoints and the tandem duplication in a head-to-tail orientation. Gastroenterology 2010 139, 2221-2224DOI: (10.1053/j.gastro.2010.08.063) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Germline epimutation of the protein tyrosine phosphatase PTPRJ gene induced by a constitutional micro-duplication. (A) Schematic representation of the head-to-tail in tandem micro-duplication encompassing exons 1–11 of the PTPRJ gene. Transcription of the duplicated segment reads through into exon 2 of the downstream intact copy of the gene, thereby inducing hypermethylation and subsequent silencing of its CpG island (CGI) promoter. (B) Reverse transcriptase PCR analysis using primers in exon 11 (P11F) and exon 3 (P3R) of the PTPRJ gene, revealing an aberrant fusion transcript (see also 2C) in lymphoblastoid cells (LC), normal colon mucosa (NC), and tumor (Tu) of the index patient (III:4) as well as in lymphoblastoid cells of her father (II:1), but not in an unaffected control (C) LC. An exon 13 to exon 14 (P13F-P14R) reverse transcriptase PCR product was used as input control. (C) Sequence analysis of the P11F-P3R reverse transcriptase PCR fragment derived from patient III:4 showing the aberrant transcript fusion between exon 11 and exon 2. (D) Sequence of PTPRJ exon 13 using lymphoblastoid cell-derived genomic DNA showing a coding SNP rs4752904 (middle), which is heterozygous in the patient (III:4) and homozygous in the father (II:1), both carriers of the duplication. Sequencing of the expressed PTPRJ transcripts revealed that only the wild-type C-allele was expressed, whereas an unrelated heterozygous control sample showed normal bi-allelic expression. (E, F) Relative levels of methylation in LC, NC, and Tu of the index patient (in duplo) and in 9 unrelated controls as determined by bisulfite sequencing of an amplified region within the CpG island of the PTPRJ promoter. For each amplicon, 20–25 independent clones were sequenced. Depicted are the percentages of clones showing <30% methylation (white bars), 30%–60% methylation (gray bars), and >60% methylation (black bars). Gastroenterology 2010 139, 2221-2224DOI: (10.1053/j.gastro.2010.08.063) Copyright © 2010 AGA Institute Terms and Conditions