The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination.

PublishedJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tAntibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are similarly initiated in B cells with the generation of U:G mismatches by activation-induced cytidine deaminase but differ in their subsequent mutagenic consequences. Although SHM relies on the generation of nondeleterious point mutations, CSR depends on the production of DNA double-strand breaks (DSBs) and their adequate recombination through nonhomologous end joining (NHEJ). MLH1, an ATPase member of the mismatch repair (MMR) machinery, is emerging as a likely regulator of whether a U:G mismatch progresses toward mutation or DSB formation. We conducted experiments on cancer modeled ATPase-deficient MLH1G67R knockin mice to determine the function that the ATPase domain of MLH1 mediates in SHM and CSR. Mlh1(GR/GR) mice displayed a significant decrease in CSR, mainly attributed to a reduction in the generation of DSBs and diminished accumulation of 53BP1 at the immunoglobulin switch regions. However, SHM was normal in these mice, which distinguishes MLH1 from upstream members of the MMR pathway and suggests a very specific role of its ATPase-dependent functions during CSR. In addition, we show that the residual switching events still taking place in Mlh1(GR/GR) mice display unique features, suggesting a role for the ATPase activity of MLH1 beyond the activation of the endonuclease functions of its MMR partner PMS2. A preference for switch junctions with longer microhomologies in Mlh1(GR/GR) mice suggests that through its ATPase activity, MLH1 also has an impact in DNA end processing, favoring canonical NHEJ downstream of the DSB. Collectively, our study shows that the ATPase domain of MLH1 is important to transmit the CSR signaling cascade both upstream and downstream of the generation of DSBs.Spanish Ministry of Education and ScienceNIHNational Women’s Division of the Albert Einstein College of Medicin

FGFR3 and PIK3CA mutations are involved in the molecular pathogenesis of solar lentigo

Solar lentigines (SL) are frequent benign skin lesions appearing on sun-exposed areas especially in elderly people and therefore represent a hallmark of (photo)aged skin. It has been proposed that SL may subsequently evolve into adenoid seborrhoeic keratosis (SK). However, little is known about the genetic basis of SL. In human SK, FGFR3 and PIK3CA mutations have recently been identified. To analyse SL for potential FGFR3 and PIK3CA mutations. We screened 30 SL for FGFR3 mutations using a SNaPshot((R)) multiplex assay. For PIK3CA mutations we used direct sequencing of exon 9 and a SNaPshot((R)) assay for the H1047R hotspot mutation (exon 20). Because psoralen plus ultraviolet A (PUVA) lentigines show the V600E BRAF hotspot mutation, we additionally investigated this mutation in SL by allele-specific polymerase chain reaction. FGFR3 mutations were detected in five of 30 (17%) SL and PIK3CA mutations in two of 28 (7%) SL. None of 28 SL available for BRAF analysis revealed the V600E mutation. Our results suggest that FGFR3 and PIK3CA mutations are involved in the pathogenesis of SL. The occurrence of these mutations in both SL and SK suggests a common genetic basis. Our findings furthermore substantiate previous speculations that UV exposure may be a causative factor for FGFR3 and PIK3CA mutations in human skin

Prediction of Progression of Non-Muscle-Invasive Bladder Cancer by WHO 1973 and 2004 Grading and by FGFR3 Mutation Status: A Prospective Study

Objectives: The clinical management of non-muscle-invasive urothelial cell carcinoma of the bladder (UCC) is challenging, as it has a marked tendency to recur and to progress. Aim of this study was to investigate the prognostic value of the WHO 1973 and 2004 grading systems and biomarkers FGFR3, CK20 and Ki-67. Methods: In a prospective study, tumours from 221 patients were studied for the expression of CK20 and Ki-67 by immunohistochemistry, and FGFR3 status by SNaPshot mutation detection. Staging and grading were performed according to the WHO classification systems of 1973 and 2004. Results: : Median follow-up was 35 mo. Recurrence occurred in 72 of 221 patients. None of the parameters was able to predict disease recurrence. CK20, Ki-67, FGFR3 mutation, molecular grade using FGFR3 mutation analysis and Ki-67, and histological grading and staging were significantly associated with disease progression in stage. in multivariable analyses, WHO 1973 and 2004 grading systems remained statistically significant and independent predictors of progression, with p = 0.005 for WHO 1973 and p = 0.004 for 2004. FGFR3 status was able to discriminate progressors from nonprogressors in a subset of patients with high-grade UCC (p = 0.009). Conclusions: This is the first prospective study comparing the WHO 1973 and 2004 grading systems. We show that both grading systems contribute valuable independent information. Therefore, it should be considered whether a better grading system could be developed that incorporates essential elements from both. The combination of WHO 2004 grading with FGFR3 status allows a better risk stratification for patients with high-grade non-muscle-invasive UCC. (C) 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved