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Allelic imbalance of the TGFβR1 is not a major contributor to the genetic predisposition to colorectal cancer.

Abadie C, Killian A, Tinat J, Bougeard M, Medhaoui D, Cailleux AF, Baert-Desurmont S, Frebourg T - Br. J. Cancer (2011)

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Whereas an allelic expression imbalance of the transforming growth factor-β type 1 receptor (TGFβR1) gene was reported in 2008 to be a strong risk factor for colorectal cancer (CRC), with an odds ratio of 8.7, this new study coordinated by Valle and performed on 96 CRC patients and 90 controls shows that there is no difference in blood TGFβR1 allelic expression between CRC patients and controls... We would like to highlight the fact that, to determine whether or not TGFβR1 allelic expression is a main CRC genetic factor conferring a high odds ratio, the selection both of patients and controls is a critical issue... Exclusion criteria were (i) Lynch syndrome defined on the basis of a germline deleterious MMR gene mutation or a CRC with microsatellite instability arising in a suggestive familial context; (ii) adenomatous polyposis defined as the presence of at least 10 colorectal adenomatous polyps... The controls, aged from 45 to 60 years, were CRC-free and had no familial history of CRC in their first-degree relatives... For each patient and control, genomic DNA was first genotyped for the following four TGFβR1 SNPs: rs334348, rs334349, rs1590 (in total linkage disequilibrium) and rs7871490, to identify heterozygous individuals... After RT–PCR was performed from total RNA using oligo-dT (New England Biolabs, Ipswich, MA, USA) and reverse transcriptase SuperScript II (Invitrogen, Cergy-Pontoise, France), allelic expression of TGFβR1 was measured, using SNaPshot, by normalising the ratio of the allelic peaks obtained on RT–PCR with the ratio of the allele peaks obtained on genomic DNA... The allelic expression was calculated from the average of the ratio obtained for the four SNPs, when individuals were informative for all these SNPs; for individuals informative only for the rs7871490 SNP, the analysis was performed on two independent RT–PCRs... Among 175 Caucasian controls and 110 Caucasian patients genotyped for the four SNPs, we measured allelic expression of TGFβR1 in 98 informative controls (56%) and 69 informative patients (63%)... Using the SNaPshot analysis, we did not encounter any reproducibility problem: for heterozygous individuals for the four SNPs (rs334348, rs334349, rs1590 and rs7871490), we obtained consistent ratio values between each SNP and, even for the rs7871490 SNP localised in a repetitive sequence, we observed reproducible results between two independent analyses (average s.d.=0.075)... Allelic expression ratios ranged between 0.82 and 1.41 (mean 1.06) in controls and between 0.77 and 1.45 (mean 1.07) in patients with the relative same compacted distribution of values in both analysed groups (see Figure 1)... No significant difference in allelic expression ratios between patients and controls was observed (Mann–Whitney test P=0.72; Student's test P=0.58; permutation test P=0.56)... In conclusion, we confirmed by studying highly selected patients and controls that allelic imbalance of the TGFβR1 is not a major contributor to the genetic predisposition to CRC.

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Ratios of TGFβR1 allelic expression in 98 informative controls and 69 patients. The median and inter-quartile range values are indicated by continuous traits. The cutoff values, indicated as discontinuous lines, were those defined according to Valle et al (2008).
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fig1: Ratios of TGFβR1 allelic expression in 98 informative controls and 69 patients. The median and inter-quartile range values are indicated by continuous traits. The cutoff values, indicated as discontinuous lines, were those defined according to Valle et al (2008).

Mentions: Therefore, we conducted a prospective case–control study on carefully selected patients and controls to clarify the real contribution of TGFβR1 allelic imbalance in CRC. After informed consent, patients were selected according to three different criteria suggestive of an increased genetic risk for CRC: (i) CRC before 61 years of age (or high-risk adenoma before 51 years of age) with a first-degree relative presenting with CRC; (ii) CRC before 51 years of age (or high-risk adenoma before 41 years of age); or (iii) multiple primitive colorectal tumours in the same case, the first one diagnosed before 61 years of age if cancer or before 51 years of age if high-risk adenoma. Exclusion criteria were (i) Lynch syndrome defined on the basis of a germline deleterious MMR gene mutation or a CRC with microsatellite instability arising in a suggestive familial context; (ii) adenomatous polyposis defined as the presence of at least 10 colorectal adenomatous polyps. The controls, aged from 45 to 60 years, were CRC-free and had no familial history of CRC in their first-degree relatives. Genomic DNA was extracted from whole blood samples using the FlexiGene kit (Qiagen, Courtaboeuf, France). As high-quality RNA is a pre-requisite to perform such a study, blood samples were collected on PAXgene tubes (Qiagen), stored at −20°C and total RNA was extracted within 1 month after blood collection, using the PAXgene Blood RNA kit (Qiagen). To measure TGFβR1 allelic expression, we used the SNaPshot methodology (Applied Biosystems, Meylan, France) described in the initial report of Valle et al (2008). For each patient and control, genomic DNA was first genotyped for the following four TGFβR1 SNPs: rs334348, rs334349, rs1590 (in total linkage disequilibrium) and rs7871490, to identify heterozygous individuals. After RT–PCR was performed from total RNA using oligo-dT (New England Biolabs, Ipswich, MA, USA) and reverse transcriptase SuperScript II (Invitrogen, Cergy-Pontoise, France), allelic expression of TGFβR1 was measured, using SNaPshot, by normalising the ratio of the allelic peaks obtained on RT–PCR with the ratio of the allele peaks obtained on genomic DNA. The allelic expression was calculated from the average of the ratio obtained for the four SNPs, when individuals were informative for all these SNPs; for individuals informative only for the rs7871490 SNP, the analysis was performed on two independent RT–PCRs. Among 175 Caucasian controls and 110 Caucasian patients genotyped for the four SNPs, we measured allelic expression of TGFβR1 in 98 informative controls (56%) and 69 informative patients (63%). Using the SNaPshot analysis, we did not encounter any reproducibility problem: for heterozygous individuals for the four SNPs (rs334348, rs334349, rs1590 and rs7871490), we obtained consistent ratio values between each SNP and, even for the rs7871490 SNP localised in a repetitive sequence, we observed reproducible results between two independent analyses (average s.d.=0.075). Allelic expression ratios ranged between 0.82 and 1.41 (mean 1.06) in controls and between 0.77 and 1.45 (mean 1.07) in patients with the relative same compacted distribution of values in both analysed groups (see Figure 1). No significant difference in allelic expression ratios between patients and controls was observed (Mann–Whitney test P=0.72; Student's test P=0.58; permutation test P=0.56).


Allelic imbalance of the TGFβR1 is not a major contributor to the genetic predisposition to colorectal cancer.

Abadie C, Killian A, Tinat J, Bougeard M, Medhaoui D, Cailleux AF, Baert-Desurmont S, Frebourg T - Br. J. Cancer (2011)

Ratios of TGFβR1 allelic expression in 98 informative controls and 69 patients. The median and inter-quartile range values are indicated by continuous traits. The cutoff values, indicated as discontinuous lines, were those defined according to Valle et al (2008).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101938&req=5

fig1: Ratios of TGFβR1 allelic expression in 98 informative controls and 69 patients. The median and inter-quartile range values are indicated by continuous traits. The cutoff values, indicated as discontinuous lines, were those defined according to Valle et al (2008).
Mentions: Therefore, we conducted a prospective case–control study on carefully selected patients and controls to clarify the real contribution of TGFβR1 allelic imbalance in CRC. After informed consent, patients were selected according to three different criteria suggestive of an increased genetic risk for CRC: (i) CRC before 61 years of age (or high-risk adenoma before 51 years of age) with a first-degree relative presenting with CRC; (ii) CRC before 51 years of age (or high-risk adenoma before 41 years of age); or (iii) multiple primitive colorectal tumours in the same case, the first one diagnosed before 61 years of age if cancer or before 51 years of age if high-risk adenoma. Exclusion criteria were (i) Lynch syndrome defined on the basis of a germline deleterious MMR gene mutation or a CRC with microsatellite instability arising in a suggestive familial context; (ii) adenomatous polyposis defined as the presence of at least 10 colorectal adenomatous polyps. The controls, aged from 45 to 60 years, were CRC-free and had no familial history of CRC in their first-degree relatives. Genomic DNA was extracted from whole blood samples using the FlexiGene kit (Qiagen, Courtaboeuf, France). As high-quality RNA is a pre-requisite to perform such a study, blood samples were collected on PAXgene tubes (Qiagen), stored at −20°C and total RNA was extracted within 1 month after blood collection, using the PAXgene Blood RNA kit (Qiagen). To measure TGFβR1 allelic expression, we used the SNaPshot methodology (Applied Biosystems, Meylan, France) described in the initial report of Valle et al (2008). For each patient and control, genomic DNA was first genotyped for the following four TGFβR1 SNPs: rs334348, rs334349, rs1590 (in total linkage disequilibrium) and rs7871490, to identify heterozygous individuals. After RT–PCR was performed from total RNA using oligo-dT (New England Biolabs, Ipswich, MA, USA) and reverse transcriptase SuperScript II (Invitrogen, Cergy-Pontoise, France), allelic expression of TGFβR1 was measured, using SNaPshot, by normalising the ratio of the allelic peaks obtained on RT–PCR with the ratio of the allele peaks obtained on genomic DNA. The allelic expression was calculated from the average of the ratio obtained for the four SNPs, when individuals were informative for all these SNPs; for individuals informative only for the rs7871490 SNP, the analysis was performed on two independent RT–PCRs. Among 175 Caucasian controls and 110 Caucasian patients genotyped for the four SNPs, we measured allelic expression of TGFβR1 in 98 informative controls (56%) and 69 informative patients (63%). Using the SNaPshot analysis, we did not encounter any reproducibility problem: for heterozygous individuals for the four SNPs (rs334348, rs334349, rs1590 and rs7871490), we obtained consistent ratio values between each SNP and, even for the rs7871490 SNP localised in a repetitive sequence, we observed reproducible results between two independent analyses (average s.d.=0.075). Allelic expression ratios ranged between 0.82 and 1.41 (mean 1.06) in controls and between 0.77 and 1.45 (mean 1.07) in patients with the relative same compacted distribution of values in both analysed groups (see Figure 1). No significant difference in allelic expression ratios between patients and controls was observed (Mann–Whitney test P=0.72; Student's test P=0.58; permutation test P=0.56).

View Article: PubMed Central - PubMed

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Whereas an allelic expression imbalance of the transforming growth factor-β type 1 receptor (TGFβR1) gene was reported in 2008 to be a strong risk factor for colorectal cancer (CRC), with an odds ratio of 8.7, this new study coordinated by Valle and performed on 96 CRC patients and 90 controls shows that there is no difference in blood TGFβR1 allelic expression between CRC patients and controls... We would like to highlight the fact that, to determine whether or not TGFβR1 allelic expression is a main CRC genetic factor conferring a high odds ratio, the selection both of patients and controls is a critical issue... Exclusion criteria were (i) Lynch syndrome defined on the basis of a germline deleterious MMR gene mutation or a CRC with microsatellite instability arising in a suggestive familial context; (ii) adenomatous polyposis defined as the presence of at least 10 colorectal adenomatous polyps... The controls, aged from 45 to 60 years, were CRC-free and had no familial history of CRC in their first-degree relatives... For each patient and control, genomic DNA was first genotyped for the following four TGFβR1 SNPs: rs334348, rs334349, rs1590 (in total linkage disequilibrium) and rs7871490, to identify heterozygous individuals... After RT–PCR was performed from total RNA using oligo-dT (New England Biolabs, Ipswich, MA, USA) and reverse transcriptase SuperScript II (Invitrogen, Cergy-Pontoise, France), allelic expression of TGFβR1 was measured, using SNaPshot, by normalising the ratio of the allelic peaks obtained on RT–PCR with the ratio of the allele peaks obtained on genomic DNA... The allelic expression was calculated from the average of the ratio obtained for the four SNPs, when individuals were informative for all these SNPs; for individuals informative only for the rs7871490 SNP, the analysis was performed on two independent RT–PCRs... Among 175 Caucasian controls and 110 Caucasian patients genotyped for the four SNPs, we measured allelic expression of TGFβR1 in 98 informative controls (56%) and 69 informative patients (63%)... Using the SNaPshot analysis, we did not encounter any reproducibility problem: for heterozygous individuals for the four SNPs (rs334348, rs334349, rs1590 and rs7871490), we obtained consistent ratio values between each SNP and, even for the rs7871490 SNP localised in a repetitive sequence, we observed reproducible results between two independent analyses (average s.d.=0.075)... Allelic expression ratios ranged between 0.82 and 1.41 (mean 1.06) in controls and between 0.77 and 1.45 (mean 1.07) in patients with the relative same compacted distribution of values in both analysed groups (see Figure 1)... No significant difference in allelic expression ratios between patients and controls was observed (Mann–Whitney test P=0.72; Student's test P=0.58; permutation test P=0.56)... In conclusion, we confirmed by studying highly selected patients and controls that allelic imbalance of the TGFβR1 is not a major contributor to the genetic predisposition to CRC.

Show MeSH