Reduced mRNA expression in paraffin-embedded tissue identifies MLH1- and MSH2-deficient colorectal tumours and potential mutation carriers
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- 作者：Annegret M¨¹ller ; Dirk Zielinski ; Nicolaus Friedrichs ; Barbara Oberschmid ; Sabine Merkelbach-Bruse ; Hans K. Schackert ; Markus Linnebacher ; Magnus von Knebel Doeberitz ; Reinhard B¨¹ttner and Josef R¨¹schoff ; et al.
- 关键词：Diagnostic tool ; MMR ; deficient tumours ; Nonsense ; mediated decay ; qRT ; PCR
- 刊名：Virchows Archiv
- 出版年：2008
- 出版时间：July, 2008
- 年：2008
- 卷：453
- 期：1
- 页码：9-16
- 全文大小：244.3 KB
文摘
Based on the principle of nonsense-mediated mRNA decay, we sought to identify MLH1 or MSH2-deficient colorectal tumours through relative quantification of mRNA expression with real-time PCR (RT-PCR) analysis. MLH1 and MSH2 mRNAs were almost equally expressed as defined by MLH1 to MSH2 transcript ratio (mean 1.41) in microsatellite stable, mismatch repair (MMR) proficient tumours (n = 16). A close correlation between loss of protein expression and MMR–mRNA levels was found in highly microsatellite instable (MSI-H) tumours deficient of MLH1 or MSH2. MLH1/MSH2 ratio was low in 11 sporadic and nine hereditary MLH1-deficient carcinomas (mean 0.51), whereas the ratio was high in 17 MSH2-deficient hereditary non-polyposis colorectal cancer (HNPCC) associated carcinomas (mean 6.8). Notably, in the normal tissues of HNPCC patients with MSH2 mutations, the MLH1/MSH2 transcript ratios were significantly elevated (ratio > 2.0) as compared to the ratios of normal mucosa in patients with MMR-proficient tumours (27 of 32 ratio < 2.0; p = 0.00113). Analysis of B-lymphocytes of HNPCC patients with proven MMR gene mutation confirmed these findings. In conclusion, RT-PCR allows relative quantification of MMR gene mRNA expression in formalin-fixed and paraffin-embedded tissue. Furthermore, this approach enables quantification of haploinsufficiency due to nonsense-mediated mRNA decay in normal tissue and B-lymphocytes from patients carrying MSH2 germline mutations and may be useful for identification of asymptomatic carriers of pathogenic germline mutations.