Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.
Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10−6 to 37.5×10−6, with a median of 4.9×10−6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.
Background: Hemodialysis with high cut-off continuous veno-venous hemodialyzer (HCO-CVVHD) removes mediators of organ dysfunction during sepsis. This study assessed the clinical effects of HCO-CVVHD as compared to high-flux (HF) membranes during gram-negative sepsis. Methods: Intensive care unit (ICU), septic patients treated with HCO-CVVHD or HF-CVVHDF for AKI were retrospectively observed (January 2013-December 2014). Mechanical ventilation, vasopressors' requirements, ICU length of stay (LOS) and ICU in-hospital mortality were compared between groups. Results: Sixteen HCO and 8 HF patients were observed. Isolated pathogens included Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa. Median ratios of days-on-vasopressors and days-on-mechanical ventilation/ICU-LOS were 0.5, 1 and 0.8, 1 for HCO and HF groups (p < 0.03), respectively. ICU-LOS was 16 and 9 days (HCO- and HF-group, p = 0.03). ICU mortality rates were 37.5 and 87.5% for HCO and HF groups, respectively (p = 0.03). No statistical difference was found in in-hospital morality. Conclusion: Patients in HCO-CVVHD group spent lesser number of days on vasopressors and mechanical ventilation as a ratio to total ICU-LOS. In the same group, a reduction in ICU mortality was observed.
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