Carol Paterson

A Multifactorial Likelihood Model for MMR Gene Variant Classification Incorporating Probabilities Based on Sequence Bioinformatics and Tumor Characteristics: A Report from the Colon Cancer Family Registry

Mismatch repair (MMR) gene sequence variants of uncertain clinical significance are often identified in suspected Lynch syndrome families, and this constitutes a challenge for both researchers and clinicians. Multifactorial likelihood model approaches provide a quantitative measure of MMR variant pathogenicity, but first require input of likelihood ratios (LRs) for different MMR variation‐associated characteristics from appropriate, well‐characterized reference datasets. Microsatellite instability (MSI) and somatic BRAF tumor data for unselected colorectal cancer probands of known pathogenic variant status were used to derive LRs for tumor characteristics using the Colon Cancer Family Registry (CFR) resource. These tumor LRs were combined with variant segregation within families, and estimates of prior probability of pathogenicity based on sequence conservation and position, to analyze 44 unclassified variants identified initially in Australasian Colon CFR families. In addition, in vitro splicing analyses were conducted on the subset of variants based on bioinformatic splicing predictions. The LR in favor of pathogenicity was estimated to be ∼12‐fold for a colorectal tumor with a BRAF mutation‐negative MSI‐H phenotype. For 31 of the 44 variants, the posterior probabilities of pathogenicity were such that altered clinical management would be indicated. Our findings provide a working multifactorial likelihood model for classification that carefully considers mode of ascertainment for gene testing.

Regulation of the Atm promoter in vivo.

While ATM, the protein defective in the human genetic disorder ataxia‐telangiectasia (A‐T), is primarily activated as a preexisting protein by radiation, there is also evidence that expression of the protein can be regulated at the transcriptional level. Activation of the ATM promoter by ionizing radiation has been reported only in quiescent cells in culture. To investigate how the Atm promoter is regulated in vivo, we generated transgenic mice that express the luciferase reporter gene under the control of the murine Atm promoter. Using a biophotonic imaging system luciferase activity was monitored in vivo. Strong promoter activity was detected throughout the transgenic animals with particularly high signals from the thymus, abdominal region, and reproductive organs. This activity further increased in response to both ionizing radiation and heat stress in a time dependent manner. Luciferase activity, measured in vitro in extracts from different tissues, showed highest activities in testes, ovaries, and cerebellum. Subjecting these mice to a single dose of 4 Gy total body radiation led to a time‐dependent activation of the promoter with the strongest response observed in the peritoneal membrane, skin, and spleen. For most tissues tested, maximal promoter activity was reached 8 hr after radiation. The observed changes in promoter activity largely correlated with levels and activity of Atm protein in tissue extracts. These results demonstrate that, in addition to activation by autophosphorylation, Atm can also be regulated in vivo at the transcriptional level possibly ensuring a more sustained response to radiation and other stimuli.

Estimation of probabilities in favour of pathogenicity for missense substitutions for use in clinical evaluation of mismatch repair gene variants

A considerable proportion of Lynch syndrome families present with mismatch repair (MMR) gene sequence variants of uncertain clinical significance, which constitute a challenge in both the research and clinical settings. Such unclassified variants (UVs) include rare nucleotide changes predicted to cause missense substitutions, small in-frame deletions, or possible alterations in splicing. We are developing a MMR multifactorial likelihood model to provide a quantitative measure of MMR variant pathogenicity. Bayes analysis of families to measure variant causality by segregation methods is established. Likelihood ratios for microsatellite instability and somatic BRAF tumour status have also recently been incorporated into the multifactorial model. We are currently estimating the prior probability of pathogenicity for MMR missense substitutions based on the evolutionary conservation and physicochemical properties of amino acid alterations. To this end, we have built and curated multiple-species sequence alignments of the four MMR proteins. In parallel, we identified 143 apparent missense substitutions, excluded 12 with evidence of causing a splice defect, and then used a combination of quantitative and qualitative criteria to classify 73 as either pathogenic, likely pathogenic, likely not pathogenic, or not pathogenic, based on the IARC five-class system. Six different missense substitution analysis tools (Align-GVGD, MAPP, MutPred, PolyPhen-2.1, SIFT, and Xvir) were used to score the missense substitutions. The bioinformatic outputs are being calibrated by regression against the classifications of the 73 missense substitutions to estimate a prior probability of pathogenicity for MMR missense substitutions. In summary, we have developed a MMR classification model including tumour characteristics, segregation analysis, and in silico prior probability of pathogenicity for missense substitutions. This multifactorial model will be applied to the analysis of 34 UVs from Australasian families, for which we have already completed assessment of tumour pathology, variant association with disease in families, and bioinformatic and in vitro splicing analysis. Results from this analysis will alter the management of these and other MMR variant-carrying families.