Oligonucleotide-directed Mutation Screening: a cellular assay to study MMR gene variants of uncertain significance

Hellen Houlleberghs, Marleen Dekker, Thomas van Ravesteyn, Chantal Stoepker, Hein te Riele, INVUSE consortium

Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. Phone +31-20-5122084; Email h.t.riele@nki.nl


Carriers of a deleterious DNA mismatch repair (MMR) gene variant (deletion, stop codon) are cancer prone (Lynch syndrome, LS) and need surveillance to reduce cancer risk. However, single codon variants are often difficult to interpret and families carrying such ‘Variants of uncertain significance’ (VUS) cannot be properly counseled as long as the functional implications of the variant remain uncertain.


We have developed a functional cellular test to study MMR gene VUS termed: “oligonucleotide-directed mutation screening” (ODMS) (Houlleberghs et al., PNAS 2016;113:4128, PLoS Genet 2017;13:e1006765). Briefly, the variant is introduced into mouse embryonic stem cells (ESCs), hemizygous for the MMR gene concerned, by oligonucleotide-directed gene modification (Van Ravesteyn et al., PNAS 2016;113:4122). This technique uses short (25 nucleotides) single-stranded oligodeoxyribonucleotides (ssODN) to introduce the VUS into the endogenous gene in ±0.01% of cultured cells. When the VUS is deleterious, modified cells are MMR deficient and survive exposure to the base analog 6-thioguanine (6TG) to form colonies. Should the VUS not affect MMR, no colonies appear. This protocol identified 64 deleterious VUS among 149 MMR gene variants.


Because of high level of conservation, MMR gene variants affecting amino acid composition can reliably be studied in mouse cells. However, to address extra-exonic variants located in less-well-conserved regions (promoter, intron and 5’- and 3’-untranslated sequences), we optimized ODMS in a human diploid cell line.