Enhanced gene targeting to evaluate Lynch syndrome alterations.

The identification of gene alterations that predispose individuals to disease has resulted in an explosion in genetic diagnostics. Because diseases like cancer susceptibility may result in debilitating surgery that has physical, emotional, and realized costs, it is important to get the diagnosis right. The obvious solution is to combine simple DNA genotype analysis with evocative phenotype analysis (1). However, a simple phenotypic analysis for the complex progressions of cancer that often includes dozens of attendant mutations (2) seemed largely destined to fail. Enter Lynch syndrome, a dominant predisposition to colorectal, endometrial, ovarian, and upper urinary tract cancers that is caused by heterozygous germ-line mutations in the mismatch repair (MMR) system (3). MMR recognizes and repairs mismatched nucleotides that principally arise from polymerase misincorporation during replication (4). When MMR is defective, spontaneous mutation rates dramatically increase, accelerating the evolution of tumors (mutator hypothesis) (5). The simplicity of Lynch syndrome cause-and-effect belies the enigma of genetic diagnostics, which has uncovered abundant missense variants of unknown significance (VUS) that pepper the entire coding regions of the core human MMR genes (Fig. 1). In PNAS, two papers provide useful solutions to the genotype–phenotype problem of Lynch syndrome diagnostics. In the first, van Ravesteyn et al. (6) developed an improved rapid gene-targeting methodology for mouse embryonic stem cells that then allowed Houlleberghs et al. (7) to advance functional analysis of Lynch syndrome VUS in mammalian cells. MMR is a relatively simple excision–resynthesis process that requires MutS homologs (MSH) to recognize the mismatch and transmit this recognition along the DNA to MutL homologs (MLH/PMS) that ultimately activate excision (4). The replication polymerase complex then completes resynthesis of the resulting ssDNA gap. In human cells, the MSH2– MSH6 heterodimer is responsible for mismatch recognition and the MLH1–PMS2 heterodimer for downstream excision activation (4). Mutation of these core MMR genes MSH2 (33%), MSH6 (18%), MLH1 (42%), and PMS2 (7%) accounts for the vast majority of Lynch syndrome cases, with most of the alterations being nonsense or frameshift mutations with obviously defective function (8). Because genetic diagnostics have become commonplace and Lynch syndrome is relatively frequent in the human population, the numbers of VUS have become significant. As a consequence, members of the International Society for Gastrointestinal Hereditary Tumors (InSiGHT) proposed guidelines to classify the disease significance of MMR gene variants (9). A five-tiered system was developed that assigned variants into a significance category based on multiple MSH2