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De Paolis E, Nero C, Micarelli E, Leoni G, Piermattei A, Trozzi R, Scarselli E, D'Alise AM, Giacò L, De Bonis M, Preziosi A, Daniele G, Piana D, Pasciuto T, Zannoni G, Minucci A, Scambia G, Urbani A, Fanfani F. Characterization of shared neoantigens landscape in Mismatch Repair Deficient Endometrial Cancer. NPJ Precis Oncol 2024; 8:283. [PMID: 39706858 DOI: 10.1038/s41698-024-00779-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Accepted: 12/11/2024] [Indexed: 12/23/2024] Open
Abstract
Endometrial cancer (EC) with Mismatch Repair deficiency (MMRd) is characterized by the accumulation of insertions/deletions at microsatellite sites. These mutations lead to the synthesis of frameshift peptides (FSPs) that represent tumor-specific neoantigens (nAg) proved to be shared across patients/tumors with MMRd. In this study, we explored the feasibility of a nAg-based cancer vaccination design in EC with MMRd. We adopted a whole exome sequencing approach and ad hoc bioinformatics pipelines to characterize FSPs in 35 patients with EC. A mean of 146 mutated mononucleotide repeats (MNRs) was identified with enrichment in the patients' group with MLH1 impairment. A high coverage emerged from the comparative analysis of the EC FSPs with the content of the previously validated NOUS-209 vaccine. We obtained pieces of evidence of FSPs translation as expressed proteins from Ribo-seq, supporting the potential as the target of vaccination. The development of a nAgs-based vaccine strategy in MMRd EC may be further explored.
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Affiliation(s)
- Elisa De Paolis
- Departmental Unit of Molecular and Genomic Diagnostics, Genomics Research Core Facility, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
- Clinical Chemistry, Biochemistry and Molecular Biology Operations (UOC), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Camilla Nero
- Department of Woman and Child's Health and Public Health Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
- Catholic University of Sacred Heart, Rome, Italy
| | | | | | - Alessia Piermattei
- Pathology Unit, Department of Woman and Child's Health and Public Health Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
| | - Rita Trozzi
- Department of Woman and Child's Health and Public Health Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
- Catholic University of Sacred Heart, Rome, Italy
| | | | | | - Luciano Giacò
- Bioinformatics Research Core Facility, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Maria De Bonis
- Departmental Unit of Molecular and Genomic Diagnostics, Genomics Research Core Facility, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Alessia Preziosi
- Bioinformatics Research Core Facility, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Gennaro Daniele
- Phase 1 Unit, Fondazione Policlinico Universitario Agostino Gemelli, IRCCS, Rome, Italy; Scientific Directorate, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
| | - Diletta Piana
- Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Catholic University of Sacred Heart, Rome, Italy
| | - Tina Pasciuto
- Research Core Facilty Data Collection, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
- Section of Hygiene, University Department of Life Sciences and Public Health, Catholic University of Sacred Heart, Rome, Italy
| | - Gianfranco Zannoni
- Pathology Unit, Department of Woman and Child's Health and Public Health Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
- Pathology Institute, Catholic University of Sacred Heart, Rome, Italy
| | - Angelo Minucci
- Departmental Unit of Molecular and Genomic Diagnostics, Genomics Research Core Facility, Gemelli Science and Technology Park (GSTeP), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Giovanni Scambia
- Department of Woman and Child's Health and Public Health Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
- Catholic University of Sacred Heart, Rome, Italy
| | - Andrea Urbani
- Clinical Chemistry, Biochemistry and Molecular Biology Operations (UOC), Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.
- Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Catholic University of Sacred Heart, Rome, Italy.
| | - Francesco Fanfani
- Department of Woman and Child's Health and Public Health Sciences, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
- Catholic University of Sacred Heart, Rome, Italy
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Munteanu CV, Marian C, Chiriță-Emandi A, Puiu M, Trifa AP. In silico splicing analysis of the PMS2 gene: exploring alternative molecular mechanisms in PMS2-associated Lynch syndrome. BMC Genom Data 2024; 25:100. [PMID: 39592919 PMCID: PMC11600730 DOI: 10.1186/s12863-024-01281-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2024] [Accepted: 11/14/2024] [Indexed: 11/28/2024] Open
Abstract
Lynch syndrome (LS) is one of the most common hereditary cancer syndrome in human populations, associated with germline variants in MLH1, MSH2/EPCAM, MSH6 and PMS2 genes. The advent of next generation sequencing has proven a significant impact in germline variant detection in the causative genes; however, a large proportion of patients with clinical criteria still receive uncertain or negative results. PMS2 is the least frequent reported gene, associated with up to 15% of LS cases with late-onset disease and low penetrance phenotype; however, the proportion of PMS2-LS cases is considered to be highly underestimated. In this context, our analysis aimed to improve the current diagnostic yield by focusing on missense and intronic PMS2 variants available in public clinical databases (ClinVar, LOVD). We performed an in silico assessment of the wild-type DNA sequence and the reported genetic variants, employing splicing bioinformatics tools known for their effectiveness in other genes. Splicing variants were predicted in silico and using GTEx short-read RNA expression data. Out of the 2384 missense variants discovered, 90% were classified with uncertain significance (VUS). 4.9% of missense variants were shown to have a potential splicing consequence (DS > 0.2) using SpliceAI. As described in the original publication, SpliceAI-visual was proven effective in annotation of short intronic variants (< 50 bp). Four short intronic variants were identified using SpliceAI-visual as potentially splicing disturbing, in spite of using a lower threshold (DS > 0.1). Exons 2, 3, 4, 5, 6, 7, 8, 11, 12 and 14 were consistently predicted in at least three out of eight software with weak canonical splice sites. Additionally, we noted that both Exonic Splicing Enhancers (ESEs) and Exonic Splicing Silencers (ESSs) contribute significantly to alternative splicing and exonic selection in PMS2 gene. Specifically, ESE motifs were consistently more abundant in highly expressed exons 5, 11 and 14, while ESS motifs played a fundamental role in exons 6, 7 and 10. Computational analysis performed in our study serves as a valuable filtering step for guiding further RNA experiments. Additional functional data is necessary to validate our findings.
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Affiliation(s)
- Cătălin Vasile Munteanu
- Doctoral School, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania.
- Regional Center of Medical Genetics Timiș, Louis Țurcanu Clinical Emergency Hospital for Children, 2 Iosif Nemoianu Street, Timișoara, 300011, Romania.
| | - Cătălin Marian
- Department of Biochemistry and Pharmacology, Biochemistry Discipline, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania
- Center for Research and Innovation in Personalized Medicine of Respiratory Diseases, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania
- Center of Expertise on Rare Pulmonary Diseases, Victor Babeș Clinical Hospital of Infectious Diseases and Pneumophysiology, 13 Gheorghe Adam Street, Timișoara, 300310, Romania
| | - Adela Chiriță-Emandi
- Department of Microscopic Morphology, Genetics Discipline, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania
- Regional Center of Medical Genetics Timiș, Louis Țurcanu Clinical Emergency Hospital for Children, 2 Iosif Nemoianu Street, Timișoara, 300011, Romania
- Center for Genomic Medicine, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timisoara, 300041, Romania
| | - Maria Puiu
- Department of Microscopic Morphology, Genetics Discipline, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania
- Regional Center of Medical Genetics Timiș, Louis Țurcanu Clinical Emergency Hospital for Children, 2 Iosif Nemoianu Street, Timișoara, 300011, Romania
- Center for Genomic Medicine, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timisoara, 300041, Romania
| | - Adrian Pavel Trifa
- Department of Microscopic Morphology, Genetics Discipline, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania
- Center for Research and Innovation in Personalized Medicine of Respiratory Diseases, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square Street, Timișoara, 300041, Romania
- Center of Expertise on Rare Pulmonary Diseases, Victor Babeș Clinical Hospital of Infectious Diseases and Pneumophysiology, 13 Gheorghe Adam Street, Timișoara, 300310, Romania
- Breast Cancer Center, The Oncology Institute "Prof. Dr. Ion Chiricuta", 34-36 Republicii Street, Cluj-Napoca, 400015, Romania
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3
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McLean ZL, Gao D, Correia K, Roy JCL, Shibata S, Farnum IN, Valdepenas-Mellor Z, Kovalenko M, Rapuru M, Morini E, Ruliera J, Gillis T, Lucente D, Kleinstiver BP, Lee JM, MacDonald ME, Wheeler VC, Mouro Pinto R, Gusella JF. Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat. Nat Commun 2024; 15:3182. [PMID: 38609352 PMCID: PMC11015039 DOI: 10.1038/s41467-024-47485-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2023] [Accepted: 03/30/2024] [Indexed: 04/14/2024] Open
Abstract
Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.
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Affiliation(s)
- Zachariah L McLean
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - Dadi Gao
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - Kevin Correia
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Jennie C L Roy
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
| | - Shota Shibata
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - Iris N Farnum
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Zoe Valdepenas-Mellor
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Marina Kovalenko
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Manasa Rapuru
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Elisabetta Morini
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
| | - Jayla Ruliera
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Tammy Gillis
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Diane Lucente
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Benjamin P Kleinstiver
- Center for Genomic Medicine and Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA
| | - Jong-Min Lee
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - Marcy E MacDonald
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - Vanessa C Wheeler
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - Ricardo Mouro Pinto
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA
| | - James F Gusella
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA.
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA, 02142, USA.
- Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, 02115, USA.
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4
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McLean ZL, Gao D, Correia K, Roy JCL, Shibata S, Farnum IN, Valdepenas-Mellor Z, Rapuru M, Morini E, Ruliera J, Gillis T, Lucente D, Kleinstiver BP, Lee JM, MacDonald ME, Wheeler VC, Pinto RM, Gusella JF. PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.07.25.550489. [PMID: 37547003 PMCID: PMC10402039 DOI: 10.1101/2023.07.25.550489] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/08/2023]
Abstract
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1, a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1, making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.
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Affiliation(s)
- Zachariah L. McLean
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
| | - Dadi Gao
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
| | - Kevin Correia
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Jennie C. L. Roy
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Shota Shibata
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
| | - Iris N. Farnum
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Zoe Valdepenas-Mellor
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Manasa Rapuru
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Elisabetta Morini
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Jayla Ruliera
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Tammy Gillis
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Diane Lucente
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Benjamin P. Kleinstiver
- Center for Genomic Medicine and Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
| | - Jong-Min Lee
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
| | - Marcy E. MacDonald
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
| | - Vanessa C. Wheeler
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Ricardo Mouro Pinto
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
| | - James F. Gusella
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
- Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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Amemiya K, Hirotsu Y, Nagakubo Y, Watanabe S, Amemiya S, Mochizuki H, Oyama T, Kondo T, Omata M. Simple IHC reveals complex MMR alternations than PCR assays: Validation by LCM and next-generation sequencing. Cancer Med 2022; 11:4479-4490. [PMID: 35596629 PMCID: PMC9741978 DOI: 10.1002/cam4.4832] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Revised: 04/07/2022] [Accepted: 05/04/2022] [Indexed: 12/15/2022] Open
Abstract
Evaluation of the status of mismatch repair (MMR) in tumors is crucial for determining the application of immune checkpoint inhibitors (ICIs). Conventional PCR (MSI-PCR) is the gold standard for confirming the MMR status. However, it requires visual confirmation and presents difficulties in determining MMR status. Immunohistochemistry (IHC) is a simple method and can confirming MMR protein expression in the whole tumor. We aim to investigate IHC is more suitable for evaluating MMR status in the tumor. We compared MSI-PCR and IHC by testing 319 samples from 284 patients across 14 cancer types. In discordant cases, we performed laser-capture microdissection and microsatellite instability assay by next-generation sequencing (MSI-NGS). The concordance rate between IHC and MSI-PCR testing was 98.1% (313/319). Two reasons for these discrepancies were ambiguous MSI-PCR results and heterogeneous MSI status within the tumor. Among six cases (1.9%), three were judged as MSI-H by MSI-PCR but with proficient MMR by IHC. The results of MSI-NGS revealed microsatellite stable in these three cases. The remaining three cases, two of three were MSI-H and one was MSS in whole tumor in MSI-PCR. IHC showed a "mosaic" pattern containing both proficient MMR and deficient MMR portions by IHC in all three cases. We performed microdissection and MSI-PCR and found intratumoral heterogeneity of MMR status. These results indicated the advantages of IHC and performed expanded samples (n = 1082) and two additional mosaic cases were identified. Our results clearly indicated that simple IHC is the best choice for determining MMR alterations in critical cases for ICIs treatment.
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Affiliation(s)
- Kenji Amemiya
- Division of Genetics and Clinical LaboratoryYamanashi Cental HospitalYamanashiJapan
- Genome Analysis CenterYamanashi Cental HospitalYamanashiJapan
- Department of Pathology, School of MedicineUniversity of YamanashiYamanashiJapan
| | - Yosuke Hirotsu
- Division of Genetics and Clinical LaboratoryYamanashi Cental HospitalYamanashiJapan
- Genome Analysis CenterYamanashi Cental HospitalYamanashiJapan
| | - Yuki Nagakubo
- Division of Genetics and Clinical LaboratoryYamanashi Cental HospitalYamanashiJapan
| | | | - Saki Amemiya
- Department of PathologyYamanashi Central HospitalYamanashiJapan
| | | | - Toshio Oyama
- Department of PathologyYamanashi Central HospitalYamanashiJapan
| | - Tetsuo Kondo
- Department of Pathology, School of MedicineUniversity of YamanashiYamanashiJapan
| | - Masao Omata
- Department of GastroenterologyYamanashi Central HospitalYamanashiJapan
- Department of GastroenterologyThe University of TokyoTokyoJapan
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6
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D'Arcy BM, Arrington J, Weisman J, McClellan SB, Vandana , Yang Z, Deivanayagam C, Blount J, Prakash A. PMS2 variant results in loss of ATPase activity without compromising mismatch repair. Mol Genet Genomic Med 2022; 10:e1908. [PMID: 35189042 PMCID: PMC9034662 DOI: 10.1002/mgg3.1908] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 02/08/2022] [Indexed: 02/01/2023] Open
Abstract
Hereditary cancer syndromes account for approximately 5%-10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N-terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in-cell fluorescent-based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign.
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Affiliation(s)
- Brandon M. D'Arcy
- Mitchell Cancer InstituteUniversity of South Alabama HealthMobileAlabamaUSA
- Department of Biochemistry and Molecular BiologyUniversity of South AlabamaMobileAlabamaUSA
| | - Jennifer Arrington
- Mitchell Cancer InstituteUniversity of South Alabama HealthMobileAlabamaUSA
- Department of Biochemistry and Molecular BiologyUniversity of South AlabamaMobileAlabamaUSA
| | - Justin Weisman
- Mitchell Cancer InstituteUniversity of South Alabama HealthMobileAlabamaUSA
- Department of Biochemistry and Molecular BiologyUniversity of South AlabamaMobileAlabamaUSA
| | - Steven B. McClellan
- Mitchell Cancer InstituteUniversity of South Alabama HealthMobileAlabamaUSA
- Flow Cytometry Shared Resource LabMitchell Cancer InstituteMobileAlabamaUSA
| | - Vandana
- Mitchell Cancer InstituteUniversity of South Alabama HealthMobileAlabamaUSA
- Department of Biochemistry and Molecular BiologyUniversity of South AlabamaMobileAlabamaUSA
| | - Zhengrong Yang
- Department of Biochemistry and Molecular GeneticsSchool of Medicine University of Alabama at BirminghamBirminghamAlabamaUSA
| | - Champion Deivanayagam
- Department of Biochemistry and Molecular GeneticsSchool of Medicine University of Alabama at BirminghamBirminghamAlabamaUSA
| | | | - Aishwarya Prakash
- Mitchell Cancer InstituteUniversity of South Alabama HealthMobileAlabamaUSA
- Department of Biochemistry and Molecular BiologyUniversity of South AlabamaMobileAlabamaUSA
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7
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Wu MT, Ye WT, Wang YC, Chen PM, Liu JY, Tai CK, Tang FY, Li JR, Liu CC, Chiang EPI. MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA. Int J Mol Sci 2021; 22:ijms22179392. [PMID: 34502300 PMCID: PMC8431311 DOI: 10.3390/ijms22179392] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 08/11/2021] [Accepted: 08/19/2021] [Indexed: 12/14/2022] Open
Abstract
Folate depletion causes chromosomal instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in the carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the partitioning of intracellular one-carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to establish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a lentiviral vector, and (3) investigated the impacts of reduced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and are critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had a worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein, and enzymatic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in wildtype (WT), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate the methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M, and cell population in the G2/M was inversely correlated with MTHFR gene level (r = −0.81, p < 0.0001), MTHFR protein expression (r = −0.8; p = 0.01), and MTHFR enzyme activity (r = −0.842; p = 0.005). Folate depletion resulted in G2/M cell cycle arrest in WT and Neg but not in shMTHFR cells, indicating that shMTHFR does not exacerbate folate depletion-induced G2/M cell cycle arrest. In addition, shMTHFR promoted the expression and translocation of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate-dependent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion-induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight into the significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies.
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Affiliation(s)
- Ming-Tsung Wu
- Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; (M.-T.W.); (W.-T.Y.); (Y.-C.W.); (P.-M.C.); (J.-Y.L.)
- Department of Civil and Environmental Engineering, South Kensington Campus, Imperial College London, London SW7 2AZ, UK
| | - Wei-Ting Ye
- Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; (M.-T.W.); (W.-T.Y.); (Y.-C.W.); (P.-M.C.); (J.-Y.L.)
| | - Yi-Cheng Wang
- Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; (M.-T.W.); (W.-T.Y.); (Y.-C.W.); (P.-M.C.); (J.-Y.L.)
| | - Po-Ming Chen
- Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; (M.-T.W.); (W.-T.Y.); (Y.-C.W.); (P.-M.C.); (J.-Y.L.)
| | - Jun-You Liu
- Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; (M.-T.W.); (W.-T.Y.); (Y.-C.W.); (P.-M.C.); (J.-Y.L.)
| | - Chien-Kuo Tai
- Department of Biomedical Sciences, National Chung Cheng University, Chia-Yi 62102, Taiwan;
| | - Feng-Yao Tang
- Department of Nutrition, China Medical University, Taichung 40402, Taiwan;
| | - Jian-Rong Li
- Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 40227, Taiwan; (J.-R.L.); (C.-C.L.)
| | - Chun-Chi Liu
- Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 40227, Taiwan; (J.-R.L.); (C.-C.L.)
| | - En-Pei Isabel Chiang
- Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan; (M.-T.W.); (W.-T.Y.); (Y.-C.W.); (P.-M.C.); (J.-Y.L.)
- Innovation and Development Center of Sustainable Agriculture (IDCSA), National Chung Hsing University, Taichung 40227, Taiwan
- Correspondence:
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8
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Yang RR, Li KKW, Zhang ZY, Chan AKY, Wang WW, Chan DTM, Li WC, Liu XZ, Li FC, Chen H, Ng HK, Mao Y, Shi ZF. Mismatch repair proteins PMS2 and MLH1 can further refine molecular stratification of IDH-mutant lower grade astrocytomas. Clin Neurol Neurosurg 2021; 208:106882. [PMID: 34428613 DOI: 10.1016/j.clineuro.2021.106882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 07/14/2021] [Accepted: 08/03/2021] [Indexed: 10/20/2022]
Abstract
The diagnostic role of Isocitrate Dehydrogenase (IDH) mutation status in adult lower grade astrocytomas was first formally presented within the WHO Classification of Tumours of the Central Nervous System (2016). IDH-mutant astrocytomas are not as common as IDH-wildtype astrocytomas but are of better prognosis. Our previous study provided an evident that IDH-mutant lower grade astrocytomas is not a homogeneous group and could be further stratified by PDGFRA amplification, CDK4 amplification and CDKN2A deletion. In this study, we detected the expressions of DNA mismatch repair (MMR) proteins (PMS2, MLH1, MSH2, MSH6) and PD-L1 by immunohistochemistry in 147 IDH-mutant lower grade astrocytomas and explored their clinical relevance. The loss of was identified in 28.6%, 1.4%, 8.8% and 13.6%, respectively. PD-L1 expression was detected in 1.4% of this cohort. Survival analysis revealed that loss of PMS2 was correlated with shorter OS (p < 0.001) and PFS (p = 0.005). Loss of PMS2 or MLH1 was associated with shorter OS (p < 0.001) and PFS (p = 0.008). In IDH-mutant lower grade astrocytomas without CDKN2A deletion, loss of PMS2 was associated with poorer OS (p < 0.001) and PFS (p = 0.001). Furthermore, among IDH-mutant lower grade astrocytomas lacking the three biomarkers (PDGFRA, CDK4 and CDKN2A), loss of PMS2 was also associated with a poorer OS (p < 0.001) and PFS (p = 0.003). Our data illustrated the potential application of MMR genes in stratification of IDH-mutant lower grade astrocytomas without PDGFRA, CDK4 and CDKN2A copy number alterations.
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Affiliation(s)
- Rui Ryan Yang
- Department of Neurosurgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, China
| | - Kay Ka-Wai Li
- Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
| | - Zhen-Yu Zhang
- Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Aden Ka-Yin Chan
- Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Wei-Wei Wang
- Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Danny Tat-Ming Chan
- Division of Neurosurgery, Department of Surgery, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Wen-Cai Li
- Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Xian-Zhi Liu
- Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Fang-Cheng Li
- Department of Neurosurgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, China
| | - Hong Chen
- Department of Pathology, Huashan Hospital, Fudan University, Shanghai, China
| | - Ho-Keung Ng
- Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
| | - Ying Mao
- Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China.
| | - Zhi-Feng Shi
- Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China.
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9
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Zhao S, Chen L, Zang Y, Liu W, Liu S, Teng F, Xue F, Wang Y. Endometrial cancer in Lynch syndrome. Int J Cancer 2021; 150:7-17. [PMID: 34398969 DOI: 10.1002/ijc.33763] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 08/05/2021] [Accepted: 08/09/2021] [Indexed: 12/11/2022]
Abstract
Lynch syndrome (LS) is an autosomal dominant inherited disease caused by germline pathogenic variants (PVs) in mismatch repair (MMR) genes. LS-associated endometrial cancer (LS-EC) is the most common extraintestinal sentinel cancer caused by germline PVs in MMR genes, including MLH1, MSH2, MSH6 and PMS2. The clinicopathologic features of LS-EC include early age of onset, lower body mass index (BMI), endometrioid carcinoma and lower uterine segment involvement. There has been significant progress in screening, diagnosis, surveillance, prevention and treatment of LS-EC. Many studies support universal screening for LS among patients with EC. Screening mainly involves a combination of traditional clinical criteria and molecular techniques, including MMR-immunohistochemistry (MMR-IHC), microsatellite instability (MSI) testing, MLH1 promoter methylation testing and gene sequencing. The effectiveness of endometrial biopsy and transvaginal ultrasound (TVS) for clinical monitoring of asymptomatic women with LS are uncertain yet. Preventive strategies include hysterectomy and bilateral salpingo-oophorectomy (BSO) as well as chemoprophylaxis using exogenous progestin or aspirin. Recent research has revealed the benefits of immunotherapy for LS-EC. The NCCN guidelines recommend pembrolizumab and nivolumab for treating patients with advanced or recurrent microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) EC.
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Affiliation(s)
- Shuangshuang Zhao
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Lingli Chen
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Yuqin Zang
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Wenlu Liu
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Shiqi Liu
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Fei Teng
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Fengxia Xue
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
| | - Yingmei Wang
- Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, Tianjin, China.,Tianjin Key Laboratory of Female Reproductive Health and Eugenics, Tianjin Medical University General Hospital, Tianjin, China
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10
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Porro A, Mohiuddin M, Zurfluh C, Spegg V, Dai J, Iehl F, Ropars V, Collotta G, Fishwick KM, Mozaffari NL, Guérois R, Jiricny J, Altmeyer M, Charbonnier JB, Pearson CE, Sartori AA. FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats. SCIENCE ADVANCES 2021; 7:7/31/eabf7906. [PMID: 34330701 PMCID: PMC8324060 DOI: 10.1126/sciadv.abf7906] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Accepted: 06/15/2021] [Indexed: 05/05/2023]
Abstract
FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
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Affiliation(s)
- Antonio Porro
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Mohiuddin Mohiuddin
- Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Christina Zurfluh
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Vincent Spegg
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Jingqi Dai
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Florence Iehl
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Virginie Ropars
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Giulio Collotta
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Keri M Fishwick
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Nour L Mozaffari
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Raphaël Guérois
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Josef Jiricny
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Matthias Altmeyer
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Jean-Baptiste Charbonnier
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Christopher E Pearson
- Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
- Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Alessandro A Sartori
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
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11
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Roy JCL, Vitalo A, Andrew MA, Mota-Silva E, Kovalenko M, Burch Z, Nhu AM, Cohen PE, Grabczyk E, Wheeler VC, Mouro Pinto R. Somatic CAG expansion in Huntington's disease is dependent on the MLH3 endonuclease domain, which can be excluded via splice redirection. Nucleic Acids Res 2021; 49:3907-3918. [PMID: 33751106 PMCID: PMC8053082 DOI: 10.1093/nar/gkab152] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 02/19/2021] [Accepted: 02/23/2021] [Indexed: 11/18/2022] Open
Abstract
Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.
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Affiliation(s)
- Jennie C L Roy
- Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
| | - Antonia Vitalo
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.,Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Marissa A Andrew
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Eduarda Mota-Silva
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Marina Kovalenko
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Zoe Burch
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Anh M Nhu
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Paula E Cohen
- Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.,Center for Reproductive Genomics, Cornell University, Ithaca, NY 14853, USA
| | - Ed Grabczyk
- Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
| | - Vanessa C Wheeler
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.,Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Ricardo Mouro Pinto
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.,Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.,Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
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12
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Hayward BE, Steinbach PJ, Usdin K. A point mutation in the nuclease domain of MLH3 eliminates repeat expansions in a mouse stem cell model of the Fragile X-related disorders. Nucleic Acids Res 2020; 48:7856-7863. [PMID: 32619224 DOI: 10.1093/nar/gkaa573] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2020] [Revised: 06/23/2020] [Accepted: 07/02/2020] [Indexed: 12/17/2022] Open
Abstract
The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases, genetic disorders that result from the expansion of a disease-specific microsatellite. In those Repeat Expansion Disease models where it has been examined, expansion is dependent on functional mismatch repair (MMR) factors, including MutLγ, a heterodimer of MLH1/MLH3, one of the three MutL complexes found in mammals and a minor player in MMR. In contrast, MutLα, a much more abundant MutL complex that is the major contributor to MMR, is either not required for expansion or plays a limited role in expansion in many model systems. How MutLγ acts to generate expansions is unclear given its normal role in protecting against microsatellite instability and while MLH3 does have an associated endonuclease activity, whether that contributes to repeat expansion is uncertain. We show here, using a gene-editing approach, that a point mutation that eliminates the endonuclease activity of MLH3 eliminates expansions in an FXD mouse embryonic stem cell model. This restricts the number of possible models for repeat expansion and supports the idea that MutLγ may be a useful druggable target to reduce somatic expansion in those disorders where it contributes to disease pathology.
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Affiliation(s)
- Bruce E Hayward
- Section on Gene Structure and Disease Laboratory of Cell and Molecular Biology National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Peter J Steinbach
- Center for Molecular Modeling, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA
| | - Karen Usdin
- Section on Gene Structure and Disease Laboratory of Cell and Molecular Biology National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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13
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DNA Mismatch Repair Gene Variants in Sporadic Solid Cancers. Int J Mol Sci 2020; 21:ijms21155561. [PMID: 32756484 PMCID: PMC7432688 DOI: 10.3390/ijms21155561] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Revised: 07/29/2020] [Accepted: 07/30/2020] [Indexed: 12/18/2022] Open
Abstract
The phenotypic effects of single nucleotide polymorphisms (SNPs) in the development of sporadic solid cancers are still scarce. The aim of this review was to summarise and analyse published data on the associations between SNPs in mismatch repair genes and various cancers. The mismatch repair system plays a unique role in the control of the genetic integrity and it is often inactivated (germline and somatic mutations and hypermethylation) in cancer patients. Here, we focused on germline variants in mismatch repair genes and found the outcomes rather controversial: some SNPs are sometimes ascribed as protective, while other studies reported their pathological effects. Regarding the complexity of cancer as one disease, we attempted to ascertain if particular polymorphisms exert the effect in the same direction in the development and treatment of different malignancies, although it is still not straightforward to conclude whether polymorphisms always play a clear positive role or a negative one. Most recent and robust genome-wide studies suggest that risk of cancer is modulated by variants in mismatch repair genes, for example in colorectal cancer. Our study shows that rs1800734 in MLH1 or rs2303428 in MSH2 may influence the development of different malignancies. The lack of functional studies on many DNA mismatch repair SNPs as well as their interactions are not explored yet. Notably, the concerted action of more variants in one individual may be protective or harmful. Further, complex interactions of DNA mismatch repair variations with both the environment and microenvironment in the cancer pathogenesis will deserve further attention.
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14
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Furman CM, Elbashir R, Alani E. Expanded roles for the MutL family of DNA mismatch repair proteins. Yeast 2020; 38:39-53. [PMID: 32652606 DOI: 10.1002/yea.3512] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Revised: 07/01/2020] [Accepted: 07/08/2020] [Indexed: 12/31/2022] Open
Abstract
The MutL family of DNA mismatch repair proteins plays a critical role in excising and repairing misincorporation errors during DNA replication. In many eukaryotes, members of this family have evolved to modulate and resolve recombination intermediates into crossovers during meiosis. In these organisms, such functions promote the accurate segregation of chromosomes during the meiosis I division. What alterations occurred in MutL homolog (MLH) family members that enabled them to acquire these new roles? In this review, we present evidence that the yeast Mlh1-Mlh3 and Mlh1-Mlh2 complexes have evolved novel enzymatic and nonenzymatic activities and protein-protein interactions that are critical for their meiotic functions. Curiously, even with these changes, these complexes retain backup and accessory roles in DNA mismatch repair during vegetative growth.
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Affiliation(s)
- Christopher M Furman
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
| | - Ryan Elbashir
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
| | - Eric Alani
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA
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15
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Xiao N, Hao S, Zhang Y, Shao Z. Roles of immune responses in the pathogenesis of immunorelated pancytopenia. Scand J Immunol 2020; 92:e12911. [PMID: 32474938 DOI: 10.1111/sji.12911] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Revised: 04/09/2020] [Accepted: 05/19/2020] [Indexed: 12/14/2022]
Abstract
Some patients with pancytopenia do not conform to any diagnostic criteria of known haematological or non-haematological diseases; however, they respond well to corticosteroid, high-dose intravenous immunoglobulin and rituximab treatment. This abnormality is termed immunorelated pancytopenia (IRP). Later studies indicated that IRP might be a kind of autoimmune disease in which T helper (Th) type 2 cell function is enhanced, resulting in the hyperfunction of B lymphocytes, which then produce excess autoantibodies that attack the bone marrow (BM) and cause cytopenia. Hypofunction of regulatory T (Treg) cells and enhanced Th17 cell function, an elevated percentage of plasmacytoid dendritic cells (pDCs) and a decreased percentage of natural killer (NK) cells help to promote the process. Moreover, increased expression of a synergistic stimulator of B lymphocytes, CD70 and the reactive overexpression of the BCR inhibitory coreceptor CD22 also support this claim. Candidate autoantigens targeted by autoantibodies on haematopoietic cell membranes have also been reported in IRP. This review is focused on studies that demonstrate the role of immune responses in the pathogenesis of IRP. Current diagnostic criteria and treatments for IRP are also referenced to provide a thorough understanding. Distinguishing IRP from idiopathic cytopenias of undetermined significance (ICUS) and other haematological disorders, for example myelodysplastic syndrome (MDS), aplastic anaemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and Evans syndrome, may help patients with pancytopenia benefit from proper treatment. Further studies are required to achieve new insight into the pathophysiology of IRP with regard to the immune system, which will be instrumental for the development of novel therapies for inhibiting disease initiation and/or progression.
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Affiliation(s)
- Na Xiao
- Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China
| | - Shanfeng Hao
- Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China
| | - Yang Zhang
- Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China
| | - Zonghong Shao
- Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China
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16
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Fraune C, Simon R, Hube-Magg C, Makrypidi-Fraune G, Kähler C, Kluth M, Höflmayer D, Büscheck F, Dum D, Luebke AM, Burandt E, Clauditz TS, Wilczak W, Sauter G, Steurer S. MMR deficiency in urothelial carcinoma of the bladder presents with temporal and spatial homogeneity throughout the tumor mass. Urol Oncol 2020; 38:488-495. [PMID: 32067846 DOI: 10.1016/j.urolonc.2019.12.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2019] [Revised: 12/06/2019] [Accepted: 12/10/2019] [Indexed: 12/24/2022]
Abstract
BACKGROUND Microsatellite instability (MSI), a hypermutator phenotype described in many cancers, has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in bladder cancer, but data on the possible extent of intratumoral heterogeneity are lacking. METHODS To study MSI heterogeneity in bladder cancer, a tissue microarray (TMA) comprising 598 muscle-invasive urothelial carcinomas of the bladder was utilized to screen for MSI by immunhistochemistry with antibodies for MLH1, PMS2, MSH2, and MSH6. RESULTS In 9 cases suspicious for MSI, MMR status was further evaluated by large section examination and polymerase chain reaction (PCR)-based analysis of microsatellites ("Bethesda panel") resulting in the identification of 5 validated MSI cases from 448 interpretable cancers (prevalence 1.1%). MMR deficiency always involved PMS2 loss, in 3 cases with additional loss or reduction of MLH1 expression. Four cancers were MSI-high and 1 was MSI-low in the PCR analysis. Parallel sequencing revealed an inactivating MLH1 mutation in 1 tumor but no further known pathogenic MMR gene mutations were found. Immunostaining of all available 72 cancer-containing tissue blocks of the 5 confirmed bladder cancer with MSI including prior and subsequent biopsies showed complete homogeneity of the MMR protein defects and the status of the 4 MMR proteins did not markedly change in sequential resections. In all 4 cases with noninvasive precursor lesions, MSI was also detectable. CONCLUSION These data suggest that MSI occurs early in invasive bladder cancer and immunohistochemical MMR analysis on limited biopsy material is sufficient to estimate MMR status of the entire cancer mass.
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Affiliation(s)
- Christoph Fraune
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Ronald Simon
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
| | - Claudia Hube-Magg
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | | | - Christian Kähler
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Martina Kluth
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Doris Höflmayer
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Franziska Büscheck
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - David Dum
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Andreas M Luebke
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Eike Burandt
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | | | - Waldemar Wilczak
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Guido Sauter
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Stefan Steurer
- Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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17
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Abildgaard AB, Stein A, Nielsen SV, Schultz-Knudsen K, Papaleo E, Shrikhande A, Hoffmann ER, Bernstein I, Gerdes AM, Takahashi M, Ishioka C, Lindorff-Larsen K, Hartmann-Petersen R. Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome. eLife 2019; 8:e49138. [PMID: 31697235 PMCID: PMC6837844 DOI: 10.7554/elife.49138] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Accepted: 10/23/2019] [Indexed: 12/13/2022] Open
Abstract
Defective mismatch repair leads to increased mutation rates, and germline loss-of-function variants in the repair component MLH1 cause the hereditary cancer predisposition disorder known as Lynch syndrome. Early diagnosis is important, but complicated by many variants being of unknown significance. Here we show that a majority of the disease-linked MLH1 variants we studied are present at reduced cellular levels. We show that destabilized MLH1 variants are targeted for chaperone-assisted proteasomal degradation, resulting also in degradation of co-factors PMS1 and PMS2. In silico saturation mutagenesis and computational predictions of thermodynamic stability of MLH1 missense variants revealed a correlation between structural destabilization, reduced steady-state levels and loss-of-function. Thus, we suggest that loss of stability and cellular degradation is an important mechanism underlying many MLH1 variants in Lynch syndrome. Combined with analyses of conservation, the thermodynamic stability predictions separate disease-linked from benign MLH1 variants, and therefore hold potential for Lynch syndrome diagnostics.
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Affiliation(s)
- Amanda B Abildgaard
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
| | - Amelie Stein
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
| | - Sofie V Nielsen
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
| | - Katrine Schultz-Knudsen
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
| | - Elena Papaleo
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
| | - Amruta Shrikhande
- DNRF Center for Chromosome Stability, Department of Cellular and Molecular MedicineUniversity of CopenhagenCopenhagenDenmark
| | - Eva R Hoffmann
- DNRF Center for Chromosome Stability, Department of Cellular and Molecular MedicineUniversity of CopenhagenCopenhagenDenmark
| | - Inge Bernstein
- Department of Surgical GastroenterologyAalborg University HospitalAalborgDenmark
| | | | - Masanobu Takahashi
- Department of Medical OncologyTohoku University Hospital, Tohoku UniversitySendaiJapan
| | - Chikashi Ishioka
- Department of Medical OncologyTohoku University Hospital, Tohoku UniversitySendaiJapan
| | - Kresten Lindorff-Larsen
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
| | - Rasmus Hartmann-Petersen
- Department of Biology, The Linderstrøm-Lang Centre for Protein ScienceUniversity of CopenhagenCopenhagenDenmark
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18
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Tamura K, Kaneda M, Futagawa M, Takeshita M, Kim S, Nakama M, Kawashita N, Tatsumi-Miyajima J. Genetic and genomic basis of the mismatch repair system involved in Lynch syndrome. Int J Clin Oncol 2019; 24:999-1011. [PMID: 31273487 DOI: 10.1007/s10147-019-01494-y] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Accepted: 06/17/2019] [Indexed: 12/11/2022]
Abstract
Lynch syndrome is a cancer-predisposing syndrome inherited in an autosomal-dominant manner, wherein colon cancer and endometrial cancer develop frequently in the family, it results from a loss-of-function mutation in one of four different genes (MLH1, MSH2, MSH6, and PMS2) encoding mismatch repair proteins. Being located immediately upstream of the MSH2 gene, EPCAM abnormalities can affect MSH2 and cause Lynch syndrome. Mismatch repair proteins are involved in repairing of incorrect pairing (point mutations and deletion/insertion of simple repetitive sequences, so-called microsatellites) that can arise during DNA replication. MSH2 forms heterodimers with MSH6 or MSH3 (MutSα, MutSβ, respectively) and is involved in mismatch-pair recognition and initiation of repair. MLH1 forms a complex with PMS2, and functions as an endonuclease. If the mismatch repair system is thoroughly working, genome integrity is maintained completely. Lynch syndrome is a state of mismatch repair deficiency due to a monoallelic abnormality of any mismatch repair genes. The phenotype indicating the mismatch repair deficiency can be frequently shown as a microsatellite instability in tumors. Children with germline biallelic mismatch repair gene abnormalities were reported to develop conditions such as gastrointestinal polyposis, colorectal cancer, brain cancer, leukemia, etc., and so on, demonstrating the need to respond with new concepts in genetic counseling. In promoting cancer genome medicine in a new era, such as by utilizing immune checkpoints, it is important to understand the genetic and genomic molecular background, including the status of mismatch repair deficiency.
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Affiliation(s)
- Kazuo Tamura
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan.
| | - Motohide Kaneda
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Mashu Futagawa
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Miho Takeshita
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Sanghyuk Kim
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Mina Nakama
- Division of Clinical Genetics, Gifu University Hospital, Gifu, Japan
| | - Norihito Kawashita
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Junko Tatsumi-Miyajima
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
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19
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Gong R, He Y, Liu XY, Wang HY, Sun LY, Yang XH, Li B, Cao XK, Ye ZL, Kong LH, Zhang DD, Li YH, Xu RH, Shao JY. Mutation spectrum of germline cancer susceptibility genes among unselected Chinese colorectal cancer patients. Cancer Manag Res 2019; 11:3721-3739. [PMID: 31118792 PMCID: PMC6500872 DOI: 10.2147/cmar.s193985] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Accepted: 02/15/2019] [Indexed: 01/20/2023] Open
Abstract
Background: Genetic factors play an important role in colorectal cancer (CRC) risk, yet the prevalence and spectrum of germline cancer susceptibility gene mutations among unselected Chinese CRC patients is largely undetermined. Methods: We performed next-generation sequencing with a 73-genes panel and analyzed the prevalence and spectrum of germline mutations in 618 unselected Chinese CRC patients. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations. Clinical characteristics were assessed by age and mutation status. Protein expressions and interactions of MLH1 missense variants were evaluated by western blot and co- immunoprecipitation. Results: Overall, 112 (18.1%) of 618 unselected Chinese CRC patients were found to carry at least one pathogenic or likely pathogenic variant (totaling 97 variants), including 70 (11.3%) Lynch syndrome (LS) mutation carriers and 42 (6.8%) non-LS mutation carriers. LS mutation carriers were significantly younger at CRC diagnosis and were more likely to have right-sided, poorly differentiated, early stage, high-frequency microsatellite instability (MSI-H) or dMMR CRC and a family history of cancer compared with noncarriers. Non-LS mutation carriers were more likely to be proficient mismatch repair (pMMR) than noncarriers (p=0.039). We found four clinical variables (gender, tumor histological stage, cancer stage and mutation status) that showed significant differences between patients younger and older than 50 years old. Higher mutation rates were found in patients under 50 years old (p=0.017). Thirty-three novel variants were discovered and evaluated as pathogenic mutations by our study. Conclusion: Given the high frequency and wide spectrum of mutations, genetic testing with a multigene panel should be considered for all Chinese CRC patients under 50 years old and is also needed to determine whether a gene is associated with CRC susceptibility and to promote clinical translation.
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Affiliation(s)
- Rui Gong
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Yuan He
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Xiao-Yun Liu
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Hai-Yun Wang
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Li-Yue Sun
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Xin-Hua Yang
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Bin Li
- Research and Development Institute of Precision Medicine, 3D Medicine Inc., Shanghai, 201114, People’s Republic of China
| | - Xin-Kai Cao
- Research and Development Institute of Precision Medicine, 3D Medicine Inc., Shanghai, 201114, People’s Republic of China
| | - Zu-Lu Ye
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
| | - Ling-Heng Kong
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Colorectal Surgery, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China
| | - Da-Dong Zhang
- Research and Development Institute of Precision Medicine, 3D Medicine Inc., Shanghai, 201114, People’s Republic of China
| | - Yu-Hong Li
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China
| | - Rui-Hua Xu
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China
| | - Jian-Yong Shao
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
- Department of Molecular Diagnostics, Sun Yat-sen University Cancer Center, Guangzhou510060, People’s Republic of China
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20
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Kasela M, Nyström M, Kansikas M. PMS2 expression decrease causes severe problems in mismatch repair. Hum Mutat 2019; 40:904-907. [PMID: 30946512 PMCID: PMC6618857 DOI: 10.1002/humu.23756] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Revised: 03/15/2019] [Accepted: 03/24/2019] [Indexed: 12/21/2022]
Abstract
PMS2 is one of the four susceptibility genes in Lynch syndrome (LS), the most common cancer syndrome in the world. Inherited mutations in DNA mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, account for approximately 90% of LS, while a relatively small number of LS families segregate a PMS2 mutation. This and the low cancer penetrance in PMS2 families suggest that PMS2 is only a moderate or low‐risk susceptibility gene. We have previously shown that even a partial expression decrease in MLH1, MSH2, or MSH6 suggests that heterozygous LS mutation carriers have MMR malfunction in constitutive tissues. Whether and how PMS2 expression decrease affects the repair capability is not known. Here, we show that PMS2 knockdown cells retaining 19%, 33%, or 53% of PMS2 expression all have significantly reduced MMR efficiency. Surprisingly, the cells retaining expression levels comparable to PMS2 mutation carriers indicate the lowest repair efficiency.
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Affiliation(s)
- Mariann Kasela
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Minna Nyström
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Minttu Kansikas
- Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
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21
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Yadav RP, Ghatak S, Chakraborty P, Lalrohlui F, Kannan R, Kumar R, Pautu JL, Zomingthanga J, Chenkual S, Muthukumaran R, Senthil Kumar N. Lifestyle chemical carcinogens associated with mutations in cell cycle regulatory genes increases the susceptibility to gastric cancer risk. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2018; 25:31691-31704. [PMID: 30209766 DOI: 10.1007/s11356-018-3080-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/16/2018] [Accepted: 08/27/2018] [Indexed: 06/08/2023]
Abstract
In the present study, we correlated the various lifestyle habits and their associated mutations in cell cycle (P21 and MDM2) and DNA damage repair (MLH1) genes to investigate their role in gastric cancer (GC). Multifactor dimensionality reduction (MDR) analysis revealed the two-factor model of oral snuff and smoked meat as the significant model for GC risk. The interaction analysis between identified mutations and the significant demographic factors predicted that oral snuff is significantly associated with P21 3'UTR mutations. A total of five mutations in P21 gene, including three novel mutations in intron 2 (36651738G > A, 36651804A > T, 36651825G > T), were identified. In MLH1 gene, two variants were identified viz. one in exon 8 (37053568A > G; 219I > V) and a novel 37088831C > G in intron 16. Flow cytometric analysis predicted DNA aneuploidy in 07 (17.5%) and diploidy in 33 (82.5%) tumor samples. The G2/M phase was significantly arrested in aneuploid gastric tumor samples whereas high S-phase fraction was observed in all the gastric tumor samples. This study demonstrated that environmental chemical carcinogens along with alteration in cell cycle regulatory (P21) and mismatch repair (MLH1) genes may be stimulating the susceptibility of GC by altering the DNA content level abnormally in tumors in the Mizo ethic population.
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Affiliation(s)
- Ravi Prakash Yadav
- Department of Biotechnology, Mizoram University, Aizawl, Mizoram, 796004, India
| | - Souvik Ghatak
- Department of Biotechnology, Mizoram University, Aizawl, Mizoram, 796004, India
| | - Payel Chakraborty
- Department of Biotechnology, Mizoram University, Aizawl, Mizoram, 796004, India
| | - Freda Lalrohlui
- Department of Biotechnology, Mizoram University, Aizawl, Mizoram, 796004, India
| | - Ravi Kannan
- Cachar Cancer Hospital and Research Centre, Silchar, Assam 788015, India
| | - Rajeev Kumar
- Cachar Cancer Hospital and Research Centre, Silchar, Assam 788015, India
| | - Jeremy L Pautu
- Mizoram State Cancer Institute, Zemabawk, Aizawl, Mizoram, 796017, India
| | - John Zomingthanga
- Department of Pathology, Civil Hospital, Aizawl, Mizoram, 796001, India
| | - Saia Chenkual
- Department of Surgery, Civil Hospital, Aizawl, Mizoram, 796001, India
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22
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Weßbecher IM, Hinrichsen I, Funke S, Oellerich T, Plotz G, Zeuzem S, Grus FH, Biondi RM, Brieger A. DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477). Mol Carcinog 2018; 57:1723-1734. [DOI: 10.1002/mc.22892] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Revised: 06/22/2018] [Accepted: 08/18/2018] [Indexed: 12/15/2022]
Affiliation(s)
- Isabel M. Weßbecher
- Medical Clinic I; Biomedical Research Laboratory; Goethe-University; Frankfurt Germany
| | - Inga Hinrichsen
- Medical Clinic I; Biomedical Research Laboratory; Goethe-University; Frankfurt Germany
| | - Sebastian Funke
- Department of Ophthalmology; Experimental Ophthalmology; University Medical Center; Gutenberg University; Mainz Germany
| | - Thomas Oellerich
- Department of Medicine II, Hematology/Oncology; Goethe-University; Frankfurt Germany
| | - Guido Plotz
- Medical Clinic I; Biomedical Research Laboratory; Goethe-University; Frankfurt Germany
| | - Stefan Zeuzem
- Medical Clinic I; Biomedical Research Laboratory; Goethe-University; Frankfurt Germany
| | - Franz H. Grus
- Department of Ophthalmology; Experimental Ophthalmology; University Medical Center; Gutenberg University; Mainz Germany
| | - Ricardo M. Biondi
- Medical Clinic I; Biomedical Research Laboratory; Goethe-University; Frankfurt Germany
- Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society; Buenos Aires Argentina
| | - Angela Brieger
- Medical Clinic I; Biomedical Research Laboratory; Goethe-University; Frankfurt Germany
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23
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Halabi A, Fuselier KTB, Grabczyk E. GAA•TTC repeat expansion in human cells is mediated by mismatch repair complex MutLγ and depends upon the endonuclease domain in MLH3 isoform one. Nucleic Acids Res 2018; 46:4022-4032. [PMID: 29529236 PMCID: PMC5934671 DOI: 10.1093/nar/gky143] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Accepted: 02/15/2018] [Indexed: 12/12/2022] Open
Abstract
DNA repeat expansion underlies dozens of progressive neurodegenerative disorders. While the mechanisms driving repeat expansion are not fully understood, increasing evidence suggests a central role for DNA mismatch repair. The mismatch repair recognition complex MutSβ (MSH2-MSH3) that binds mismatched bases and/or insertion/deletion loops has previously been implicated in GAA•TTC, CAG•CTG and CGG•CCG repeat expansion, suggesting a shared mechanism. MutSβ has been studied in a number of models, but the contribution of subsequent steps mediated by the MutL endonuclease in this pathway is less clear. Here we show that MutLγ (MLH1-MLH3) is the MutL complex responsible for GAA•TTC repeat expansion. Lentiviral expression of shRNA targeting MutL nuclease components MLH1, PMS2, and MLH3 revealed that reduced expression of MLH1 or MLH3 reduced the repeat expansion rate in a human Friedreich ataxia cell model, while targeting PMS2 did not. Using splice-switching oligonucleotides we show that MLH3 isoform 1 is active in GAA•TTC repeat expansion while the nuclease-deficient MLH3 isoform 2 is not. MLH3 isoform switching slowed repeat expansion in both model cells and FRDA patient fibroblasts. Our work indicates a specific and active role for MutLγ in the expansion process and reveals plausible targets for disease-modifying therapies.
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Affiliation(s)
- Anasheh Halabi
- Division of Neurology, Department of Neurosciences, University of California, San Diego, CA 92103, USA
| | - Kayla T B Fuselier
- Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
| | - Ed Grabczyk
- Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
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24
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Hinrichsen I, Weßbecher IM, Huhn M, Passmann S, Zeuzem S, Plotz G, Biondi RM, Brieger A. Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2. Mol Carcinog 2017; 56:2663-2668. [DOI: 10.1002/mc.22709] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2017] [Accepted: 07/28/2017] [Indexed: 11/12/2022]
Affiliation(s)
- Inga Hinrichsen
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Isabel M. Weßbecher
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Meik Huhn
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
- Pharmazentrum Frankfurt; Institute of Pharmacology and Toxicology; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Sandra Passmann
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Stefan Zeuzem
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Guido Plotz
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Ricardo M. Biondi
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
| | - Angela Brieger
- Medical Clinic I; Biomedical Research Laboratory; University Clinic Frankfurt; Frankfurt a.M. Germany
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25
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Fujii N. Potential Strategies to Target Protein-Protein Interactions in the DNA Damage Response and Repair Pathways. J Med Chem 2017; 60:9932-9959. [PMID: 28654754 DOI: 10.1021/acs.jmedchem.7b00358] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This review article discusses some insights about generating novel mechanistic inhibitors of the DNA damage response and repair (DDR) pathways by focusing on protein-protein interactions (PPIs) of the key DDR components. General requirements for PPI strategies, such as selecting the target PPI site on the basis of its functionality, are discussed first. Next, on the basis of functional rationale and biochemical feasibility to identify a PPI inhibitor, 26 PPIs in DDR pathways (BER, MMR, NER, NHEJ, HR, TLS, and ICL repair) are specifically discussed for inhibitor discovery to benefit cancer therapies using a DNA-damaging agent.
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Affiliation(s)
- Naoaki Fujii
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital , 262 Danny Thomas Place, MS1000, Memphis, Tennessee 38105, United States
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26
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Yu W, Wang Z, Fong C, Liu D, Yip T, Au S, Zhu G, Yang M. Chemoresistant lung cancer stem cells display high DNA repair capability to remove cisplatin-induced DNA damage. Br J Pharmacol 2017; 174:302-313. [PMID: 27933604 PMCID: PMC5289946 DOI: 10.1111/bph.13690] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Revised: 10/23/2016] [Accepted: 11/05/2016] [Indexed: 01/26/2023] Open
Abstract
BACKGROUND AND PURPOSE The persistence of lung cancer stem cells (LCSCs) has been proposed to be the main factor responsible for the recurrence of lung cancer as they are highly resistant to conventional chemotherapy. However, the underlying mechanisms are still unclear. EXPERIMENTAL APPROACH We examined the cellular response of a human LCSC line to treatment with cisplatin, a DNA-damaging anticancer drug that is used extensively in the clinic. We compared the response to cisplatin of LCSCs and differentiated LCSCs (dLCSCs) by determining the viability of these cells, and their ability to accumulate cisplatin and to implement genomic and transcription-coupled DNA repair. We also investigated the transcription profiles of genes related to drug transport and DNA repair. KEY RESULTS LCSCs were found to be more stem-like, and more resistant to cisplatin-induced cytotoxicity than dLCSCs, confirming their drug resistance properties. LCSCs accumulated less cisplatin intracellularly than dLCSCs and showed less DNA damage, potentially due to their ability to down-regulate AQP2 and CTR1. The results of the transcription-coupled repair of cisplatin-DNA cross-links indicated a higher level of repair of DNA damage in LCSCs than in dLCSCs. In addition, LCSCs showed a greater ability to repair cisplatin-DNA interstrand cross-links than dLCSCs; this involved the activation of various DNA repair pathways. CONCLUSIONS AND IMPLICATIONS Our results further clarify the mechanism of cisplatin resistance in LCSCs in terms of reduced cisplatin uptake and enhanced ability to implement DNA repairs. These findings may aid in the design of the next-generation of platinum-based anticancer drugs.
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Affiliation(s)
- Wai‐Kin Yu
- Department of Biomedical SciencesCity University of Hong KongKowloonHong Kong
| | - Zhigang Wang
- Shenzhen Key Laboratory of Biochip ResearchCity University of Hong Kong Shenzhen Research InstituteShenzhenChina
- Department of Biology and ChemistryCity University of Hong KongKowloonHong Kong
| | - Chi‐Chun Fong
- Department of Biomedical SciencesCity University of Hong KongKowloonHong Kong
- Shenzhen Key Laboratory of Biochip ResearchCity University of Hong Kong Shenzhen Research InstituteShenzhenChina
| | - Dandan Liu
- Department of Biomedical SciencesCity University of Hong KongKowloonHong Kong
| | - Tak‐Chun Yip
- Department of Clinical OncologyQueen Elizabeth HospitalYau Ma TeiHong Kong
| | - Siu‐Kie Au
- Department of Clinical OncologyQueen Elizabeth HospitalYau Ma TeiHong Kong
| | - Guangyu Zhu
- Shenzhen Key Laboratory of Biochip ResearchCity University of Hong Kong Shenzhen Research InstituteShenzhenChina
- Department of Biology and ChemistryCity University of Hong KongKowloonHong Kong
| | - Mengsu Yang
- Department of Biomedical SciencesCity University of Hong KongKowloonHong Kong
- Shenzhen Key Laboratory of Biochip ResearchCity University of Hong Kong Shenzhen Research InstituteShenzhenChina
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Hu MH, Liu SY, Wang N, Wu Y, Jin F. Impact of DNA mismatch repair system alterations on human fertility and related treatments. J Zhejiang Univ Sci B 2016; 17:10-20. [PMID: 26739522 DOI: 10.1631/jzus.b1500162] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
DNA mismatch repair (MMR) is one of the biological pathways, which plays a critical role in DNA homeostasis, primarily by repairing base-pair mismatches and insertion/deletion loops that occur during DNA replication. MMR also takes part in other metabolic pathways and regulates cell cycle arrest. Defects in MMR are associated with genomic instability, predisposition to certain types of cancers and resistance to certain therapeutic drugs. Moreover, genetic and epigenetic alterations in the MMR system demonstrate a significant relationship with human fertility and related treatments, which helps us to understand the etiology and susceptibility of human infertility. Alterations in the MMR system may also influence the health of offspring conceived by assisted reproductive technology in humans. However, further studies are needed to explore the specific mechanisms by which the MMR system may affect human infertility. This review addresses the physiological mechanisms of the MMR system and associations between alterations of the MMR system and human fertility and related treatments, and potential effects on the next generation.
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Affiliation(s)
- Min-hao Hu
- Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China
| | - Shu-yuan Liu
- Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China
| | - Ning Wang
- Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China
| | - Yan Wu
- Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China
| | - Fan Jin
- Key Laboratory of Reproductive Genetics (Zhejiang), Ministry of Education, and Centre of Reproductive Medicine, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China
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Single-nucleotide polymorphism rs 175080 in the MLH3 gene and its relation to male infertility. J Assist Reprod Genet 2015; 32:1795-9. [PMID: 26520453 DOI: 10.1007/s10815-015-0594-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Accepted: 10/02/2015] [Indexed: 10/22/2022] Open
Abstract
PURPOSE MLH3, a MutL homolog protein in mammals playing a role in DNA mismatch repair, is associated with spermatogenesis and male infertility. The purpose of the present study was to investigate the association of the single-nucleotide polymorphism (SNP), rs 175080 in the MLH3 gene, with sperm parameters in a Greek population. METHODS The study included 300 men of couples undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments (years 2011-2013). Genomic DNA was extracted from 300 peripheral blood samples, and conventional quantitative real-time PCR was performed for genotyping. Of them, 122 were from men used as "controls" and 178 from men used as "cases." Allocation to the two groups was based on sperm concentrations (≥15 and <15 million/ml, respectively). Serum FSH, LH, estradiol, testosterone, and prolactin concentrations as well as sperm parameters were compared between three genotypes (GG, GA, and AA). Furthermore, the frequencies of these three genotypes were compared between "cases" and "controls." RESULTS Anthropometric parameters and hormonal values did not differ significantly between the three genotypes. Significantly lower sperm concentrations were found in men with the AA genotype as compared to men with the GG and GA genotypes (p < 0.001). The AA genotype had the lower progressive motility values as compared to the other two genotypes (p < 0.05). Also, there was a significantly different distribution of the frequencies of the three genotypes between "cases" and "controls" (p < 0.001). CONCLUSIONS It is suggested that the studied SNP in the MLH3 gene may be linked to oligozoospermia in Caucasian men of a certain area.
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Yamaguchi J, Sato Y, Kita M, Nomura S, Yamamoto N, Kato Y, Ishikawa Y, Arai M. A novel deletion in the splice donor site of MLH1 exon 6 in a Japanese colon cancer patient with Lynch syndrome. Jpn J Clin Oncol 2015; 45:993-7. [PMID: 26185136 DOI: 10.1093/jjco/hyv103] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2015] [Accepted: 06/13/2015] [Indexed: 01/30/2023] Open
Abstract
Lynch syndrome is an autosomal dominantly inherited disease that is characterized by a predisposition to cancers, mainly colorectal cancer. Germline mutations of DNA mismatch repair genes such as MLH1, MSH2, MSH6 and PMS2 have been described in patients with Lynch syndrome. Here, we report deletion of 2 bp in the splice donor site of the MLH1 exon 6 (c.545+4_545+5delCA) in a 48-year-old Japanese woman with Lynch syndrome. RT-PCR direct sequencing analysis revealed that this mutation led to an increase in the level of an MLH1 transcript in which exon 6 was skipped, and may cause a frameshift (p.E153FfsX8). Therefore, this mutation appears to be pathogenic and is responsible for Lynch syndrome. Additionally, analysis of the patient's tumor cells indicated microsatellite instability high phenotype and loss of the MLH1 and PMS2 proteins. To our knowledge, this is a germline splice site mutation of MLH1 that has not been reported previously.
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Affiliation(s)
- Junya Yamaguchi
- Clinical Genetic Oncology, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo
| | - Yuri Sato
- Clinical Genetic Oncology, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo
| | - Mizuho Kita
- Clinical Genetic Oncology, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo
| | - Sachio Nomura
- Clinical Genetic Oncology, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo Department of Clinical Research, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo
| | - Noriko Yamamoto
- Division of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Yo Kato
- Division of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Yuichi Ishikawa
- Division of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Masami Arai
- Clinical Genetic Oncology, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo
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Muro Y, Sugiura K, Mimori T, Akiyama M. DNA mismatch repair enzymes: Genetic defects and autoimmunity. Clin Chim Acta 2015; 442:102-9. [DOI: 10.1016/j.cca.2015.01.014] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Revised: 01/17/2015] [Accepted: 01/17/2015] [Indexed: 12/31/2022]
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Thompson B, Martins A, Spurdle A. A review of mismatch repair gene transcripts: issues for interpretation of mRNA splicing assays. Clin Genet 2014; 87:100-8. [DOI: 10.1111/cge.12450] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Revised: 06/17/2014] [Accepted: 06/24/2014] [Indexed: 12/21/2022]
Affiliation(s)
- B.A. Thompson
- Department of Genetics and Computational Biology; QIMR Berghofer Medical Research Institute; Brisbane Australia
- School of Medicine; University of Queensland; Brisbane Australia
| | - A. Martins
- Inserm U1079; University of Rouen, Institute for Research and Innovation in Biomedicine; Rouen France
| | - A.B. Spurdle
- Department of Genetics and Computational Biology; QIMR Berghofer Medical Research Institute; Brisbane Australia
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Hinrichsen I, Ernst BP, Nuber F, Passmann S, Schäfer D, Steinke V, Friedrichs N, Plotz G, Zeuzem S, Brieger A. Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1. Mol Cancer 2014; 13:11. [PMID: 24456667 PMCID: PMC3904401 DOI: 10.1186/1476-4598-13-11] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2013] [Accepted: 01/17/2014] [Indexed: 01/13/2023] Open
Abstract
Introduction Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. Methods Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. Results MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. Conclusions These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | - Angela Brieger
- Medical Clinic I, Biomedical Research Laboratory, Goethe-University, Frankfurt a,M, Germany.
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Genetic analysis of mlh3 mutations reveals interactions between crossover promoting factors during meiosis in baker's yeast. G3-GENES GENOMES GENETICS 2013; 3:9-22. [PMID: 23316435 PMCID: PMC3538346 DOI: 10.1534/g3.112.004622] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/05/2012] [Accepted: 10/30/2012] [Indexed: 12/01/2022]
Abstract
Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.
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Pradhan MP, Prasad NKA, Palakal MJ. A systems biology approach to the global analysis of transcription factors in colorectal cancer. BMC Cancer 2012; 12:331. [PMID: 22852817 PMCID: PMC3539921 DOI: 10.1186/1471-2407-12-331] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2011] [Accepted: 06/21/2012] [Indexed: 02/08/2023] Open
Abstract
Background Biological entities do not perform in isolation, and often, it is the nature and degree of interactions among numerous biological entities which ultimately determines any final outcome. Hence, experimental data on any single biological entity can be of limited value when considered only in isolation. To address this, we propose that augmenting individual entity data with the literature will not only better define the entity’s own significance but also uncover relationships with novel biological entities. To test this notion, we developed a comprehensive text mining and computational methodology that focused on discovering new targets of one class of molecular entities, transcription factors (TF), within one particular disease, colorectal cancer (CRC). Methods We used 39 molecular entities known to be associated with CRC along with six colorectal cancer terms as the bait list, or list of search terms, for mining the biomedical literature to identify CRC-specific genes and proteins. Using the literature-mined data, we constructed a global TF interaction network for CRC. We then developed a multi-level, multi-parametric methodology to identify TFs to CRC. Results The small bait list, when augmented with literature-mined data, identified a large number of biological entities associated with CRC. The relative importance of these TF and their associated modules was identified using functional and topological features. Additional validation of these highly-ranked TF using the literature strengthened our findings. Some of the novel TF that we identified were: SLUG, RUNX1, IRF1, HIF1A, ATF-2, ABL1, ELK-1 and GATA-1. Some of these TFs are associated with functional modules in known pathways of CRC, including the Beta-catenin/development, immune response, transcription, and DNA damage pathways. Conclusions Our methodology of using text mining data and a multi-level, multi-parameter scoring technique was able to identify both known and novel TF that have roles in CRC. Starting with just one TF (SMAD3) in the bait list, the literature mining process identified an additional 116 CRC-associated TFs. Our network-based analysis showed that these TFs all belonged to any of 13 major functional groups that are known to play important roles in CRC. Among these identified TFs, we obtained a novel six-node module consisting of ATF2-P53-JNK1-ELK1-EPHB2-HIF1A, from which the novel JNK1-ELK1 association could potentially be a significant marker for CRC.
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Affiliation(s)
- Meeta P Pradhan
- School of Informatics, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202, USA
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Moon DC, Choi CH, Lee SM, Lee JH, Kim SI, Kim DS, Lee JC. Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene. PLoS One 2012; 7:e38974. [PMID: 22685614 PMCID: PMC3369853 DOI: 10.1371/journal.pone.0038974] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2012] [Accepted: 05/14/2012] [Indexed: 12/18/2022] Open
Abstract
Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), 225RKRKRK230. Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.
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Affiliation(s)
- Dong Chan Moon
- Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea
| | - Chul Hee Choi
- Department of Periodontology, University of Florida, Gainesville, Florida, United States of America
| | - Su Man Lee
- Department of Anatomy, Kyungpook National University School of Medicine, Daegu, Korea
| | - Jung Hwa Lee
- Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea
| | - Seung Il Kim
- Division of Life Science, Korea Basic Science Institute, Daejeon, Korea
| | - Dong Sun Kim
- Department of Anatomy, Kyungpook National University School of Medicine, Daegu, Korea
- * E-mail: (JCL); (DSK)
| | - Je Chul Lee
- Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea
- * E-mail: (JCL); (DSK)
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Ferrás C, Fernandes S, Silva J, Barros A, Sousa M. Expression analysis of MLH3, MLH1, and MSH4 in maturation arrest. Reprod Sci 2012; 19:587-96. [PMID: 22344730 DOI: 10.1177/1933719111428521] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The expression of DNA mismatch repair (DMMR) genes in patients with maturation arrest (MA) was analyzed. Samples were subjected to mutL homolog 3 (MLH3) mutation analysis by denaturing high-performance liquid chromatography/sequencing and quantification of MMR expression in testicular tissue by real-time polymerase chain reaction (PCR). Microsatellite instability assays were negative. Two missense and 1 intronic mutations were found. The missense mutation 2531C/T (P844 L), predicted to affect MLH3 function, was found in 3 MA cases in association with the intronic variant IVS9 + 66G/A. Relative messenger RNA (mRNA) quantification identified 2 patients who overexpressed MLH3, 1 of them also overexpressing mutL homolog 1 (MLH1). The latter also presented the 2531C/T-IVS9 + 66G/A mutation. In conclusion, we suggest that a predominance of MLH3 expression might favor the MLH1/MLH3 complex which then would compete with the MLH1/PMS2 complexes. This could convey disruption of the relative stoichiometry between MLH1/MLH3 and MLH1/PMS2 complexes, thus causing meiosis failure, as MLH1/PMS2 complexes are supposed to replace MLH1/MLH3 during diplonema.
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Affiliation(s)
- Cristina Ferrás
- Laboratory of Chromosome Instability and Dynamics, Institute for Molecular Cell Biology (IBMC), Rua do Campo Alegre, Porto, Portugal
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Fan Y, Chen J, Wang W, Wu P, Zhi W, Xue B, Zhang W, Wang Y. Influence of eight unclassified missense variants of the MLH1 gene on Lynch syndrome susceptibility. Biochem Genet 2011; 50:84-93. [PMID: 21952876 DOI: 10.1007/s10528-011-9467-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2010] [Accepted: 06/02/2011] [Indexed: 01/26/2023]
Abstract
Missense mutations in MLH1 have frequently been detected in patients with Lynch syndrome, but their genetic significance has not been extensively assessed. In this study, we attempt to evaluate the etiological role of eight MLH1 missense variants. The variants were analyzed for their ability to affect MLH1 protein interaction with its partner PMS2 in vivo employing a yeast two-hybrid system. In addition, a SIFT (sorting intolerant from tolerant) algorithm was adopted to predict the effects of amino acid substitutions. Finally, scanning of mutations in a normal Chinese population and assay of the clinical characteristics have all been taken into account. Our results demonstrated that the MLH1 variants D485E and L653R cause functional alterations of the human MutLα complex significantly. The R265C, D304V, A586P, and R755S variants affect partial interaction. The remaining two variants, N38D and L559R, could be nonfunctional polymorphisms or might affect the mismatch repair system through other mechanisms.
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Affiliation(s)
- Yimei Fan
- Department of Medical Genetics, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, Jiangsu, China
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Yamamoto T, Iino H, Kim K, Kuramitsu S, Fukui K. Evidence for ATP-dependent structural rearrangement of nuclease catalytic site in DNA mismatch repair endonuclease MutL. J Biol Chem 2011; 286:42337-42348. [PMID: 21953455 DOI: 10.1074/jbc.m111.277335] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
DNA mismatch repair (MMR) greatly contributes to genome integrity via the correction of mismatched bases that are mainly generated by replication errors. Postreplicative MMR excises a relatively long tract of error-containing single-stranded DNA. MutL is a widely conserved nicking endonuclease that directs the excision reaction to the error-containing strand of the duplex by specifically nicking the daughter strand. Because MutL apparently exhibits nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of MutL has been argued. Recent studies suggest ATP-dependent conformational and functional changes of MutL, indicating that the regulatory mechanism involves the ATP binding and hydrolysis cycle. In this study, we investigated the effect of ATP binding on the structure of MutL. First, a cross-linking experiment confirmed that the N-terminal ATPase domain physically interacts with the C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass spectrometry clarified that the binding of ATP to the N-terminal domain induces local structural changes at the catalytic sites of MutL C-terminal domain. Finally, on the basis of the results of the hydrogen/deuterium exchange experiment, we successfully identified novel regions essential for the endonuclease activity of MutL. The results clearly show that ATP modulates the nicking endonuclease activity of MutL via structural rearrangements of the catalytic site. In addition, several Lynch syndrome-related mutations in human MutL homolog are located in the position corresponding to the newly identified catalytic region. Our data contribute toward understanding the relationship between mutations in MutL homolog and human disease.
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Affiliation(s)
- Tatsuya Yamamoto
- RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
| | - Hitoshi Iino
- RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
| | - Kwang Kim
- Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
| | - Seiki Kuramitsu
- RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan; Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
| | - Kenji Fukui
- RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
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Abbà S, Vallino M, Daghino S, Di Vietro L, Borriello R, Perotto S. A PLAC8-containing protein from an endomycorrhizal fungus confers cadmium resistance to yeast cells by interacting with Mlh3p. Nucleic Acids Res 2011; 39:7548-63. [PMID: 21672957 PMCID: PMC3177179 DOI: 10.1093/nar/gkr336] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Cadmium is a genotoxic pollutant known to target proteins that are involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing mutagenic and carcinogenic effects. We have identified a PLAC8 domain-containing protein, named OmFCR, by a yeast functional screen aimed at identifying genes involved in cadmium resistance in the endomycorrhizal fungus Oidiodendron maius. OmFCR shows a remarkable specificity in mediating cadmium resistance. Both its function and its nuclear localization in yeast strictly depend on the interaction with Mlh3p, a subunit of the mismatch repair (MMR) system. Although proteins belonging to the PLAC8 family are widespread in eukaryotes, they are poorly characterized and their biological role still remains elusive. Our work represents the first report about the potential role of a PLAC8 protein in physically coupling DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways. On the basis of cell survival assays and yeast growth curves, we hypothesize that, upon cadmium exposure, OmFCR might promote a higher rate of cell division as compared to control cells.
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Affiliation(s)
- S Abbà
- Dipartimento di Biologia Vegetale dell'Università degli Studi di Torino, Viale Mattioli 25, Torino, Italy.
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Schorzman AN, Perera L, Cutalo-Patterson JM, Pedersen LC, Pedersen LG, Kunkel TA, Tomer KB. Modeling of the DNA-binding site of yeast Pms1 by mass spectrometry. DNA Repair (Amst) 2011; 10:454-65. [PMID: 21354867 PMCID: PMC3084373 DOI: 10.1016/j.dnarep.2011.01.010] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2010] [Revised: 01/07/2011] [Accepted: 01/24/2011] [Indexed: 11/26/2022]
Abstract
Mismatch repair (MMR) corrects replication errors that would otherwise lead to mutations and, potentially, various forms of cancer. Among several proteins required for eukaryotic MMR, MutLα is a heterodimer comprised of Mlh1 and Pms1. The two proteins dimerize along their C-terminal domains (CTDs), and the CTD of Pms1 houses a latent endonuclease that is required for MMR. The highly conserved N-terminal domains (NTDs) independently bind DNA and possess ATPase active sites. Here we use two protein footprinting techniques, limited proteolysis and oxidative surface mapping, coupled with mass spectrometry to identify amino acids involved along the DNA-binding surface of the Pms1-NTD. Limited proteolysis experiments elucidated several basic residues that were protected in the presence of DNA, while oxidative surface mapping revealed one residue that is uniquely protected from oxidation. Furthermore, additional amino acids distributed throughout the Pms1-NTD were protected from oxidation either in the presence of a non-hydrolyzable analog of ATP or DNA, indicating that each ligand stabilizes the protein in a similar conformation. Based on the recently published X-ray crystal structure of yeast Pms1-NTD, a model of the Pms1-NTD/DNA complex was generated using the mass spectrometric data as constraints. The proposed model defines the DNA-binding interface along a positively charged groove of the Pms1-NTD and complements prior mutagenesis studies of Escherichia coli and eukaryotic MutL.
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Affiliation(s)
- Allison N. Schorzman
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
| | - Lalith Perera
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
| | - Jenny M. Cutalo-Patterson
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
| | - Lars C. Pedersen
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
| | - Lee G. Pedersen
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
| | - Thomas A. Kunkel
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
| | - Kenneth B. Tomer
- Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
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Zaanan A, Meunier K, Sangar F, Fléjou JF, Praz F. Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications. Cell Oncol (Dordr) 2011; 34:155-76. [PMID: 21484480 DOI: 10.1007/s13402-011-0024-x] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/13/2011] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Microsatellite instability (MSI) constitutes an important oncogenic molecular pathway in colorectal cancer (CRC), representing approximately 15% of all colorectal malignant tumours. In roughly one third of the cases, the underlying DNA mismatch repair (MMR) defect is inherited through the transmission of a mutation in one of the genes involved in MMR, predominantly MSH2 and MLH1, or less frequently, MSH6 or PMS2. In the overwhelming number of sporadic cases, MSI results from epigenetic MLH1 silencing through hypermethylation of its promoter. MMR deficiency promotes colorectal oncogenesis through the accumulation of numerous mutations in crucial target genes harbouring mononucleotide repeats, notably in those involved in the control of cell proliferation and differentiation, as well as DNA damage signalling and repair. DESIGN In this review, we describe the molecular aspects of the MMR system and the biological consequences of its defect on the oncogenic process, and we discuss the various experimental systems used to evaluate the efficacy of cytotoxic drugs on MSI colorectal cells lines. There is increasing evidence showing that MSI CRCs differ from all CRCs in terms of prognosis and response to the treatment. We report the clinical studies that have evaluated the prognostic and predictive value of MSI status on clinical outcome in patients treated with various chemotherapy regimens used in the adjuvant setting or for advanced CRCs. CONCLUSION In view of this, the opportunity of a systematic MSI phenotyping in the clinical management of patients with CRC is further discussed.
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Affiliation(s)
- Aziz Zaanan
- INSERM, UMR_S, Centre de Recherche Saint-Antoine, Paris, France
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Moussa SAB, Moussa A, Kourda N, Mezlini A, Abdelli N, Zerimech F, Najjar T, Jilani SB, Porchet N, Ayed FB, Manai M, Buisine MP. Lynch syndrome in Tunisia: first description of clinical features and germline mutations. Int J Colorectal Dis 2011; 26:455-67. [PMID: 21311894 DOI: 10.1007/s00384-010-1129-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 12/29/2010] [Indexed: 02/04/2023]
Abstract
PURPOSE High rates of early colorectal cancers (CRC) are observed in Tunisia suggesting genetic susceptibility. Nevertheless, up to now, no molecular study has been performed in the Tunisian population. In our research, we evaluated the clinical characteristics of Tunisian families suspected of Lynch syndrome and the contribution of DNA mismatch repair (MMR) genes. METHODS Thirty-one unrelated families suspected of Lynch syndrome were studied. Probands were tested for the presence of germline mutations in the MMR genes MLH1, MSH2, MSH6 and in MUTYH. Available tumours were analysed for microsatellite instability and expression of MMR proteins. Detailed family and medical histories were collected. RESULTS A total of 134 cancers were noted in the 31 families, the most frequent type of cancer corresponding to CRC (69%), followed by uterine cancer (7.5%). Germline mutations were identified in 11 (35.5%) families (six MSH2, five MLH1, including seven novel mutations), seven of which fulfilled the Amsterdam criteria (sensitivity, 63.6%; positive predictive value, 58.3%). Noteworthy, germline mutations were detected in 52.6% of male patients tested, but in only 8.3% of females (p = 0.02). Moreover, CRC were essentially left sided in families without detected mutation (p = 0.017). Ages of onset of cancers and tumour spectrum were very similar in families with or without MMR germline mutation, contrasting with previous studies performed in other populations. CONCLUSIONS MMR genes contribute significantly to CRC susceptibility in the Tunisian population. However, the cause of early CRC susceptibility remains unknown in most cases, especially in women and in patients with early left colon or rectal cancer.
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Affiliation(s)
- Sana Aissi-Ben Moussa
- Laboratoire de Biochimie et Biologie Moléculaire de Faculté des Sciences de Tunis, Tunis, Tunisia
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Hardt K, Heick SB, Betz B, Goecke T, Yazdanparast H, Küppers R, Servan K, Steinke V, Rahner N, Morak M, Holinski-Feder E, Engel C, Möslein G, Schackert HK, von Knebel Doeberitz M, Pox C, Hegemann JH, Royer-Pokora B. Missense variants in hMLH1 identified in patients from the German HNPCC consortium and functional studies. Fam Cancer 2011; 10:273-84. [DOI: 10.1007/s10689-011-9431-4] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Offer SM, Pan-Hammarström Q, Hammarström L, Harris RS. Unique DNA repair gene variations and potential associations with the primary antibody deficiency syndromes IgAD and CVID. PLoS One 2010; 5:e12260. [PMID: 20805886 PMCID: PMC2923613 DOI: 10.1371/journal.pone.0012260] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2010] [Accepted: 07/17/2010] [Indexed: 01/02/2023] Open
Abstract
BACKGROUND Despite considerable effort, the genetic factors responsible for >90% of the antibody deficiency syndromes IgAD and CVID remain elusive. To produce a functionally diverse antibody repertoire B lymphocytes undergo class switch recombination. This process is initiated by AID-catalyzed deamination of cytidine to uridine in switch region DNA. Subsequently, these residues are recognized by the uracil excision enzyme UNG2 or the mismatch repair proteins MutSalpha (MSH2/MSH6) and MutLalpha (PMS2/MLH1). Further processing by ubiquitous DNA repair factors is thought to introduce DNA breaks, ultimately leading to class switch recombination and expression of a different antibody isotype. METHODOLOGY/PRINCIPAL FINDINGS Defects in AID and UNG2 have been shown to result in the primary immunodeficiency hyper-IgM syndrome, leading us to hypothesize that additional, potentially more subtle, DNA repair gene variations may underlie the clinically related antibody deficiencies syndromes IgAD and CVID. In a survey of twenty-seven candidate DNA metabolism genes, markers in MSH2, RAD50, and RAD52 were associated with IgAD/CVID, prompting further investigation into these pathways. Resequencing identified four rare, non-synonymous alleles associated with IgAD/CVID, two in MLH1, one in RAD50, and one in NBS1. One IgAD patient carried heterozygous non-synonymous mutations in MLH1, MSH2, and NBS1. Functional studies revealed that one of the identified mutations, a premature RAD50 stop codon (Q372X), confers increased sensitivity to ionizing radiation. CONCLUSIONS Our results are consistent with a class switch recombination model in which AID-catalyzed uridines are processed by multiple DNA repair pathways. Genetic defects in these DNA repair pathways may contribute to IgAD and CVID.
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Affiliation(s)
- Steven M. Offer
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America
| | - Qiang Pan-Hammarström
- Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden
| | - Lennart Hammarström
- Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden
| | - Reuben S. Harris
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America
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Cantor SB, Xie J. Assessing the link between BACH1/FANCJ and MLH1 in DNA crosslink repair. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2010; 51:500-507. [PMID: 20658644 DOI: 10.1002/em.20568] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2023]
Abstract
FANCJ (also known as BRIP1 or BACH1) is a DNA helicase that was originally identified by its direct interaction with the hereditary breast cancer protein, BRCA1. Similar to BRCA1, FANCJ function is essential for DNA repair and breast cancer suppression. FANCJ is also mutated in the cancer prone syndrome Fanconi anemia, for which patient cells are characterized by extreme sensitivity to agents that generate DNA interstand crosslinks. Unexpectedly, correction of the interstrand crosslink sensitivity of FANCJ-null patient cells did not require the FANCJ/BRCA1 interaction. Instead, FANCJ binding to the mismatch repair protein, MLH1 was required. Given this finding, we address the role of FANCJ and MLH1 in DNA crosslink processing and how their functions could be linked in checkpoint and/or recombination pathways. We speculate that after DNA crosslink processing and repair, the FANCJ/MLH1 interaction is critical for recovery and restart of replication. These ideas are considered and summarized in this review.
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Affiliation(s)
- Sharon B Cantor
- Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
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Distinct regulation of Mlh1p heterodimers in meiosis and mitosis in Saccharomyces cerevisiae. Genetics 2010; 185:459-67. [PMID: 20382827 DOI: 10.1534/genetics.110.116806] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.
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A novel interaction between human DNA polymerase η and MutLα. Biochem Biophys Res Commun 2009; 389:40-5. [DOI: 10.1016/j.bbrc.2009.08.090] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2009] [Accepted: 08/13/2009] [Indexed: 01/09/2023]
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Gwin K, Wilcox R, Montag A. Insights into selected genetic diseases affecting the female reproductive tract and their implication for pathologic evaluation of gynecologic specimens. Arch Pathol Lab Med 2009; 133:1041-52. [PMID: 19642731 DOI: 10.5858/133.7.1041] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/12/2009] [Indexed: 11/06/2022]
Abstract
CONTEXT Recent advances in the understanding of genetic conditions involving the female genital tract and mechanisms of carcinogenesis in this setting affect patient management and thus necessitate appropriate pathologic evaluation of specimens. In the past, specimens from prophylactic surgery were a rarity; however, they are now more frequently encountered and often require a significant variation from routine processing methods. Pathologists also receive more specimens requiring prospective workup for possible underlying genetic conditions such as microsatellite instability. OBJECTIVE To summarize the current knowledge of important genetic and hereditary conditions affecting the female reproductive organs while highlighting the resulting practical significance for specimen handling, "grossing," and microscopic evaluation in gynecologic pathology. DATA SOURCES This update is based on a review of recent peer-reviewed literature and the experience with cases at the parent institutions. CONCLUSIONS Gynecologic specimens received from patients with certain genetic conditions require specific clinicopathologic knowledge for appropriate pathologic examination. The evaluation of prophylactic resection specimens focuses on the detection of cancer precursors and possible occult disease, which may require a more thorough and detailed examination than an obvious carcinoma. Standardized protocols for handling prophylactic gynecologic resection specimens are available for some, but not all, types of specimens. The prospective evaluation of a gynecologic pathology specimen for potential genetic conditions such as microsatellite instability is a very recent subject. Currently, well-established protocols are not available; however, as clinical and prognostic significance has become more clearly elucidated, familiarity with this evolving field is increasingly important to properly assess these pathologic specimens.
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Affiliation(s)
- Katja Gwin
- Department of Pathology, University of Chicago, Chicago, Illinois 60637-1470, USA.
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Charbonneau N, Amunugama R, Schmutte C, Yoder K, Fishel R. Evidence that hMLH3 functions primarily in meiosis and in hMSH2-hMSH3 mismatch repair. Cancer Biol Ther 2009; 8:1411-20. [PMID: 19483466 DOI: 10.4161/cbt.8.14.8886] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The MutS (MSH) and MutL (MLH) homologs are conserved proteins that function in mismatch repair (MMR) and meiosis. We examined mRNA and protein expression of hMLH3 compared to other human MSH and MLH in a panel of human tissues and the HeLa cell line. Quantitative PCR suggests that MSH and MLH transcripts are expressed ubiquitously. hMLH3 mRNA is present at low levels in numerous tissues. Protein expression appears to correlate with a threshold of mRNA expression with hMLH3 present at high levels in testis. In addition, we have found and mapped interactions between hMLH1 and hMLH3 with hMSH3. These data are consistent with yeast studies and suggest a role for hMLH3 in meiosis as well as hMSH2-hMSH3 repair processes and little if any role in Hereditary Non-Polyposis Colorectal Cancer (HNPCC).
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Affiliation(s)
- Nicole Charbonneau
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, OH, USA
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Ou J, Rasmussen M, Westers H, Andersen SD, Jager PO, Kooi KA, Niessen RC, Eggen BJL, Nielsen FC, Kleibeuker JH, Sijmons RH, Rasmussen LJ, Hofstra RMW. Biochemical characterization of MLH3 missense mutations does not reveal an apparent role of MLH3 in Lynch syndrome. Genes Chromosomes Cancer 2009; 48:340-50. [PMID: 19156873 DOI: 10.1002/gcc.20644] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
So far 18 MLH3 germline mutations/variants have been identified in familial colorectal cancer cases. Sixteen of these variants are amino acid substitutions of which the pathogenic nature is still unclear. These substitutions are known as unclassified variants or UVs. To clarify a possible role for eight of these MLH3 UVs identified in suspected Lynch syndrome patients, we performed several biochemical tests. We determined the protein expression and stability, protein localization and interaction of the mutant MLH3 proteins with wildtype MLH1. All eight MLH3 UVs gave protein expression levels comparable with wildtype MLH3. Furthermore, the UV-containing proteins, in contrast to previous studies, were all localized normally in the nucleus and they interacted normally with wildtype MLH1. Our different biochemical assays yielded no evidence that the eight MLH3 UVs tested are the cause of hereditary colorectal cancer, including Lynch syndrome.
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Affiliation(s)
- Jianghua Ou
- Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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