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Science 321 (5894): 1361-1365

Copyright © 2008 by the American Association for the Advancement of Science

Germline Allele-Specific Expression of TGFBR1 Confers an Increased Risk of Colorectal Cancer

Laura Valle1, Tarsicio Serena-Acedo1, Sandya Liyanarachchi1, Heather Hampel1, Ilene Comeras1, Zhongyuan Li1, Qinghua Zeng2, Hong-Tao Zhang2, Michael J. Pennison2, Maureen Sadim2, Boris Pasche2*, Stephan M. Tanner1*, and Albert de la Chapelle1*

1 Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
2 Cancer Genetics Program, Division of Hematology/Oncology, Department of Medicine and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

Figure 1 Fig. 1.. TGFBR1 ASE distribution in 138 CRC patients and 105 controls studied by SNaPshot. The ASE cutoff value of 1.5 chosen to categorize the cases is indicated, together with its associated P value obtained from comparing the proportions of cases (29/138) and controls (3/105) above the indicated value. [View Larger Version of this Image (17K GIF file)]

Figure 2 Fig. 2.. ASE determination in two ASE CRC probands. (A) ASE detection in blood DNA by SNaPshot. The ASE ratio was calculated by normalizing the ratio between the peak areas of the two alleles in cDNA with the same parameters in genomic DNA (gDNA). In both examples, the transcript from the a allele is reduced with respect to that from the b allele. (B) Semiquantitative RT-PCR of the cDNA from monochromosomal hybrids of the same two patients. Human TGFBR1 expression (amplicon size 135 bp) was assessed and mouse Gpi was used as a control (176 bp). The values shown below the gel represent the ratios of the densitometric values of human TGFBR1 versus mouse Gpi, showing reduced expression of human TGFBR1 in the hybrids that contain the a allele. [View Larger Version of this Image (19K GIF file)]

Figure 3 Fig. 3.. Analysis of SMAD-mediated TGF-β signaling in lymphoblastoid cell lines from ASE CRC patients and non-ASE healthy controls. (A) SMAD2 and phosphorylated SMAD2 (pSMAD2) expression were assessed by Western blotting in lymphoblastoid cell lines from ASE patients (P-1, P-5, and P-14) and non-ASE controls (C-1, C-2, and C-3), after exposure to TGF-β (100 pM) at various time points from 0 to 16 hours (h) and using β-actin as a loading control. In all three ASE cases, less constitutive pSMAD2 was observed than in non-ASE controls. The differences in pSMAD2 expression between ASE and non-ASE cell lines were further enhanced after exposure to TGF-β.(B) SMAD2 and p-SMAD2 expression 1 hour after exposure to different TGF-β concentrations. The effect shown in (A) also occurs at low concentrations of TGF-β (5 pM). (C) pSMAD3 detection in nuclear extracts from three ASE patients and three non-ASE controls after exposure to TGF-β1. The three non-ASE lymphoblastoid cell lines had pSMAD3 expression in the nucleus, whereas nuclear pSMAD3 expression was undetectable in two ASE cases (P-1 and P-14) and barely detectable in one case (P-5). [View Larger Version of this Image (71K GIF file)]

Figure 4 Fig. 4.. (A) Diagram of the TGFBR1 genomic region. The uppermost line depicts the 96.5-kb region sequenced in six ASE patients (four monochromosomal hybrids and four diploid DNAs). Shown are the locations of the 2-bp CA deletion upstream of exon 1, the 9A/6A polymorphism in exon 1, and the four SNPs in the 3'UTR used for ASE determinations. (B) Locations of the 60 SNPs used for haplotype inference in ASE (n = 31) and non-ASE (n = 55) CRC patients. The arrowed shorter lines each depict a 10-SNP overlapping window. P values indicate the significance of differences in haplotype distribution between ASE and non-ASE individuals. (C) Two major haplotypes identified in ASE patients are shown. [View Larger Version of this Image (18K GIF file)]

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