PURPOSE: Constitutional mismatch repair deficiency (CMMRD) is a highly penetrant cancer predisposition syndrome caused by biallelic mutations in mismatch repair (MMR) genes. As several cancer syndromes are clinically similar, accurate diagnosis is critical to cancer screening and treatment. As genetic diagnosis is confounded by 15 or more pseudogenes and variants of uncertain significance, a robust diagnostic assay is urgently needed. We sought to determine whether an assay that directly measures MMR activity could accurately diagnose CMMRD. PATIENTS AND METHODS: In vitro MMR activity was quantified using a 3'-nicked G-T mismatched DNA substrate, which requires MSH2-MSH6 and MLH1-PMS2 for repair. We quantified MMR activity from 20 Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with confirmed CMMRD. We also tested 20 lymphoblastoid cell lines from patients who were suspected for CMMRD. We also characterized MMR activity from patients with neurofibromatosis type 1, Li-Fraumeni syndrome, polymerase proofreading-associated cancer syndrome, and Lynch syndrome. RESULTS: All CMMRD cell lines had low MMR activity (n = 20; mean, 4.14 ± 1.56%) relative to controls (n = 6; mean, 44.00 ± 8.65%; P < .001). Repair was restored by complementation with the missing protein, which confirmed MMR deficiency. All cases of patients with suspected CMMRD were accurately diagnosed. Individuals with Lynch syndrome (n = 28), neurofibromatosis type 1 (n = 5), Li-Fraumeni syndrome (n = 5), and polymerase proofreading-associated cancer syndrome (n = 3) had MMR activity that was comparable to controls. To accelerate testing, we measured MMR activity directly from fresh lymphocytes, which yielded results in 8 days. CONCLUSION: On the basis of the current data set, the in vitro G-T repair assay was able to diagnose CMMRD with 100% specificity and sensitivity. Rapid diagnosis before surgery in non-neoplastic tissues could speed proper therapeutic management.

Aga Khan University Hospital Karachi Pakistan

Ann and Robert H Lurie Children's Hospital Northwestern University Feinberg School of Medicine Chicago IL

Banner MD Anderson Cancer Center Gilbert AZ

Baylor College of Medicine and Texas Children's Hospital Houston TX

Children Cancer Hospital Karachi Pakistan

Children's Health Dallas TX

Children's Hospital Colorado Aurora CO

Children's Hospital of Philadelphia and University of Pennsylvania Philadelphia PA

Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center Pittsburgh PA

Cleveland Clinic Weston FL

Dalhousie University Faculty of Medicine Halifax Nova Scotia Canada

Geisinger Medical Center Danville PA

King Fahad Medical City Riyadh Saudi Arabia

McGill University Health Centre Montréal Quebec Canada

McGill University Montréal Quebec Canada

Medical College of Wisconsin Milwaukee WI

Medical University of South Carolina Charleston SC

Mount Sinai Hospital Toronto Ontario Canada

Ottawa Hospital Research Institute Ottawa Ontario Canada

Princess Margaret Cancer Centre Toronto Ontario Canada

Rambam Health Care Campus Haifa Israel

Ruby Memorial Hospital Morgantown WV

Saint George Hospital University Medical Center Beirut Lebanon

Schneider Children's Medical Center of Israel Petach Tikva Israel

Sheba Medical Center Tel Hashomer Israel

Taipei Veterans General Hospital Taipei Republic of China

Tel Aviv Sourasky Medical Center Tel Aviv Israel

Tel Aviv University Tel Aviv Israel

The Hospital for Sick Children Toronto Ontario Canada

Toronto General Hospital Toronto Ontario Canada

Universidad San Francisco de Quito Quito Ecuador

Université Catholique de Louvain Brussels Belgium

Université Laval Quebec City Quebec Canada

University Hospital Motol Charles University Prague Czech Republic

University of Colorado Anschutz Medical Campus Aurora CO

University of Manitoba Winnipeg Manitoba Canada

University of Texas Southwestern Medical Center Dallas TX

University of Toronto Toronto Ontario Canada

Valley Children's Hospital Madera CA

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