doi: 10.1111/j.1469-1809.2010.00603.x MLH1 Differential Allelic Expression in Mutation Carriers and Controls Mauro Santibanez Koref 1+∗ , Valerie Wilson 2∗ , Nicola Cartwright 3 , Michael S. Cunnington 1 , John C. Mathers 3 , D. Timothy Bishop 4 , Ann Curtis 2 , Malcolm G. Dunlop 5 and John Burn 1,2 1 Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, UK 2 Northern Genetics Services, The Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK 3 Human Nutrition Research Centre, Institute of Ageing and Health, University of Newcastle, Newcastle upon Tyne, UK 4 Section of Epidemiology and Biostatistics, Leeds Institute of Molecular Medicine, University of Leeds, UK 5 Colon Cancer Genetics Group, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Summary Germline defects in the MLH1 gene are associated with Lynch syndrome. A substantial proportion of these mutations leads to premature termination codons and can induce nonsense mediated decay (NMD) of the corresponding transcript. Resulting allelic expression differences represent a fast and inexpensive method to identify patients carrying MLH1 mutations. In patients and controls, we show that allelic expression imbalance (AEI) can be readily detected in RNA extracted from whole blood from patients carrying mutations expected to elicit NMD using mass spectrometry. Mutations closer to the 5 ′ end of the gene tend to show smaller imbalances. AEI can also be detected in normal controls. Analysis of allelic expression in controls and individuals with mutations not expected to exhibit NMD revealed that MLH1 expression is influenced by sequence variation acting in cis. A maximum likelihood framework was used to identify two SNPs, rs1799977 (c.655G>A; p.I219V) and rs1800734 (c.-93 G>A) that are independently associated with expression. These influences are, however, small compared to the differences associated with pathological variants. Keywords: Allelic expression, MLH1, colon cancer, mutation, cis acting Introduction Mutations in the mismatch repair (MMR) gene MLH1, are among the most frequent causes of Lynch syndrome (Hereditary Non-Polyposis Colon Cancer HNPCC, OMIM 120435)(Peltomaki & Vasen, 1997), an autosomal dominant syndrome that accounts for approximately 5% of all colorectal cancers (Bocker et al., 1999). Pathogenic variants in other components of the MMR system, MSH2, MSH6, and PMS2 account for most other cases. MLH1 and MSH2 are the most frequently implicated (Peltomaki & Vasen, 2004; Woods et al., 2007). Lifetime risk for mutation carriers of developing can- cer was originally overestimated, raising the question of po- tential modifiers of penetrance and suggesting that a larger ∗ These authors have equal contribution. +Corresponding author: M. Santibanez Koref, Institute of Hu- man Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. Tel. +44 191 241 8696; Fax: +44 191 241 8666; E-mail: mauro.santibanez-koref@ncl.ac.uk proportion of mutations in these MMR genes may be ex- pected to occur in cases without a family history (Terdiman, 2005). This should lead to an increase in demand for mutation screening (Terdiman, 2005). The situation is complicated by the realization that a substantial proportion (30%) of mutations may not be detectable using routine sequencing (Casey et al., 2005), while epigenetic inactivation of one allele can cause cancer predisposition and may even be transmissible through the germline (Suter et al., 2004; Hitchins et al., 2007; Valle et al., 2007). Approximately 50% of known mutations in MLH1 and MSH2 result in premature termination of the encoded pro- teins, and may trigger nonsense mediated decay (NMD), the preferential degradation of mRNA molecules containing a mutation that leads to premature termination (Weischenfeldt et al., 2005; Amrani et al., 2006; Behm-Ansmant & Izaurralde, 2006). Assessing clinical utility requires knowl- edge of the extent of physiological variation. Expression lev- els of MLH1 have been described to vary by a factor of four Annals of Human Genetics (2010) 74,479–488 479 C  2010 The Authors Annals of Human Genetics C  2010 Blackwell Publishing Ltd/University College London