 With the advent of personalized genomics, the incidence of variants of unknown significance, or VUSE, in disease-predisposing genes, is increasing. In the absence of standardized procedures to assess the pathogenicity of VUSE, targeted healthcare cannot be implemented for carriers and their relatives. Lynch syndrome is a prevalent, inherited predisposition to colorectal and other visceral cancers that is caused by a defect in one of the genes of the DNA mismatch repair pathway. Disrepair removes inadvertent misincorporations introduced in DNA during its replication. Here the replicative DNA polymerase has misincorporated a guanine instead of an adenine, opposite a thymidine. In wild-type cells, the guanine-thymidine mismatch is bound by the heterodimeric MSH2-MSH6 mismatch repair protein, after which the heterodimeric emulates one PMS2 mismatch repair protein is recruited. This protein complex initiates degradation of the misincorporation containing DNA strand, which is followed by resynthesis by the DNA polymerase, this time hopefully without making a mistake. In Lynch syndrome, a pathogenic defect in one of the four DNA mismatch repair genes results in loss of mismatch repair. This leads to the persistence of the misincorporations and therefore an accumulation of spontaneous mutations, including a hallmark phenotype called microsatellite instability or MSI. These phenotypes cause a dramatically increased rate of cancer development in Lynch syndrome. The profound knowledge on the genetics, cell biology and biochemistry of DNA mismatch repair and the availability of essays allows to investigate the functional activity of the pathway as a whole and of its actors. Diagnosis of Lynch syndrome is based on clinical criteria and the search for a pathogenic defect in one of the mismatch repair genes. This results with a pathogenic defect greatly benefit from surveillance and also may benefit from both targeted lifestyle measurements and treatment. In case a vuse is identified in one of the DNA mismatch repair genes of which pathogenicity cannot be determined, all family members, whether they carry the vuse or not, remain on the surveillance. This puts a great burden on the entire family and on preventive healthcare systems. Here we propose the development of a standardized diagnostic pipeline for vuse mismatch repair genes. This pipeline is based on the causal relation between defects in DNA mismatch repair and Lynch syndrome, on the profound knowledge of the genetics, cell biology and biochemistry of DNA mismatch repair and on the availability of essays to test the activity of the DNA mismatch repair pathway as a whole and of individual components of the pathway. To assess whether a vuse is pathogenic, the diagnostic pipeline proceeds to step two. In this step, the vuse is first analyzed by computer algorithms that are based on comparative analysis of the DNA mismatch repair genes from a wide range of species. Step two also comprises a novel, cell-free essay that measures the integral activity of the DNA mismatch repair pathway in a test tube. The cell-free DNA mismatch repair essay does not require patient material, is performed entirely in a test tube and is exemplified here for testing vuse in the MSH2-M6 DNA mismatch repair protein. The variant gene and its encoded protein are synthesized in vitro. Next, the variant protein is incubated with an extract from cells that are deficient for MSH2 and MSH6 and also with a fluorescent DNA substrate that contains a mismatch. Active mismatch repair results in the creation of a restriction enzyme cleavage site which can be quantified by automated fragment analysis. Loss of DNA mismatch repair activity is characterized by the absence of the diagnostic fragment and this provides strong evidence for pathogenicity of the vuse. In some instances, the step one and two essays may not provide conclusive results as to the pathogenicity of the vuse. In these cases, the diagnostic pipeline will proceed to step three. In step three, an additional layer of diagnostic essays can include an examination of other cell biological, biochemical and biophysical analyses of the DNA mismatch repair genes and their proteins. These essays often address more specific aspects of the protein function beyond measuring repair of mismatches. The step three essays require specialized expertise, laboratory facilities and trained scientific personnel. One example of a step three essay shown here, this essay measures correct cellular localization of the variant protein. As shown here, the variant MLH1 protein does not enter the nucleus unlike the normal protein. Therefore, this result supports pathogenicity of the variant. Step three essays cannot stand alone but they can provide additional evidence in favor or against pathogenicity in case the results from the step one and two essays are inconclusive. So how can we use the results from the step one, two and three analyses to define pathogenicity of a vuse in one of the mismatch repair genes? We propose to generate a software-based hierarchical decision tree that, based on the results from the different analyses, guides the clinical geneticist through a diagnostic pipeline. The software will evaluate and integrate the essay results and perform a Beezian analysis that outputs a pathogenicity probability score ranging from not pathogenic to definitely pathogenic. This will allow the clinician to make rationalized decisions as to the management of the affected individual and his or her relatives. DNA mismatch repair is one of the best studied DNA repair pathways and biochemical defects in this pathway are causal of link syndrome. These are available to study the activity of the entire DNA mismatch repair pathway and of its components. These essays provide a unique opportunity to develop standardized diagnostic pipeline for the analysis of the pathogenicity of vuse in mismatch repair genes. Such a diagnostic procedure will not only greatly benefit individuals suspected of link syndrome and their relatives but, moreover, provide an example for the translation of genomic data into targeted healthcare in the upcoming age of personalized genomics.