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Blesa S, Olivares MD, Alic AS, Serrano A, Lendinez V, González-Albert V, Olivares L, Martínez-Hervás S, Juanes JM, Marín P, Real JT, Navarro B, García-García AB, Chaves FJ, Ivorra C. Easy One-Step Amplification and Labeling Procedure for Copy Number Variation Detection. Clin Chem 2020; 66:463-473. [PMID: 32068788 DOI: 10.1093/clinchem/hvaa002] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Accepted: 10/21/2020] [Indexed: 12/17/2022]
Abstract
BACKGROUND The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.
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Affiliation(s)
- Sebastián Blesa
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain
| | - María D Olivares
- I+D+I Department, Sequencing Multiplex SL (I+d+I, Seqplexing), Serra, Valencian Community, Spain
| | - Andy S Alic
- I+D+I Department, Sequencing Multiplex SL (I+d+I, Seqplexing), Serra, Valencian Community, Spain
| | - Alicia Serrano
- Hematology Department, Clinical University Hospital of Valencia (HCUV), Valencia, Valencian Community, Spain.,Physiology Department, University of Valencia (FD, UV), Valencia, Valencian Community, Spain
| | - Verónica Lendinez
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain
| | - Verónica González-Albert
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain
| | - Laura Olivares
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain
| | - Sergio Martínez-Hervás
- Endocrinology Service, Clinical University Hospital of Valencia (HCUV), Valencia, Valencian Community, Spain
| | - José M Juanes
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain
| | - Pablo Marín
- Computational and Clinical Genomics Department, Kanteron Systems SLU (CCGD, Kanteron), Valencia, Valencian Community, Spain
| | - Jose T Real
- Endocrinology Service, Clinical University Hospital of Valencia (HCUV), Valencia, Valencian Community, Spain.,Department of Medicine, University of Valencia (DM; UV), Valencia, Valencian Community, Spain
| | - Blanca Navarro
- Hematology Department, Clinical University Hospital of Valencia (HCUV), Valencia, Valencian Community, Spain.,Physiology Department, University of Valencia (FD, UV), Valencia, Valencian Community, Spain
| | - Ana B García-García
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain.,CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM), Madrid, Autonomous Community of Madrid, Spain
| | - Felipe J Chaves
- Genomic and Genetic Diagnosis Unit, INCLIVA Biomedical Research Institute (UGDG, INCLIVA), Valencia, Valencian Community, Spain.,I+D+I Department, Sequencing Multiplex SL (I+d+I, Seqplexing), Serra, Valencian Community, Spain.,CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM), Madrid, Autonomous Community of Madrid, Spain
| | - Carmen Ivorra
- I+D+I Department, Sequencing Multiplex SL (I+d+I, Seqplexing), Serra, Valencian Community, Spain
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Uncovering pseudotemporal trajectories with covariates from single cell and bulk expression data. Nat Commun 2018; 9:2442. [PMID: 29934517 PMCID: PMC6015076 DOI: 10.1038/s41467-018-04696-6] [Citation(s) in RCA: 77] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Accepted: 05/17/2018] [Indexed: 12/29/2022] Open
Abstract
Pseudotime algorithms can be employed to extract latent temporal information from cross-sectional data sets allowing dynamic biological processes to be studied in situations where the collection of time series data is challenging or prohibitive. Computational techniques have arisen from single-cell ‘omics and cancer modelling where pseudotime can be used to learn about cellular differentiation or tumour progression. However, methods to date typically implicitly assume homogeneous genetic, phenotypic or environmental backgrounds, which becomes limiting as data sets grow in size and complexity. We describe a novel statistical framework that learns how pseudotime trajectories can be modulated through covariates that encode such factors. We apply this model to both single-cell and bulk gene expression data sets and show that the approach can recover known and novel covariate-pseudotime interaction effects. This hybrid regression-latent variable model framework extends pseudotemporal modelling from its most prevalent area of single cell genomics to wider applications. Cross-sectional omic data often have non-homogeneous genetic, phenotypic, or environmental backgrounds. Here, the authors develop a statistical framework to infer pseudotime trajectories in the presence of such factors as well as their interactions in both single-cell and bulk gene expression analysis
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3
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Zheng J, Huang B, Nie X, Zhu Y, Han N, Li Y. The clinicopathological features and prognosis of tumor MSI in East Asian colorectal cancer patients using NCI panel. Future Oncol 2018; 14:1355-1364. [PMID: 29366338 DOI: 10.2217/fon-2017-0662] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
AIM To compare test results obtained from a PCR assay for the National Cancer Institute (NCI) five loci criteria for detecting microsatellite instability (MSI) with those obtained from immunohistochemistry of mismatch repair and a five-mononucleotide site amplification system in East Asian patients with colorectal cancer. PATIENTS & METHODS A total of 245 East Asian patients with colorectal cancer were studied retrospectively at our institution. RESULTS The consistency of the NCI panel PCR method compared with detection of mismatch repair protein expression by immunohistochemistry was 0.898. High level MSI (MSI-H) status was correlated with the Tumor, Node, Metastasis stage, tumor location site, metastasis, tumor grade, mucinous histological type and BRAF-type mutations. CONCLUSION The NCI panel PCR assay has excellent sensitivity and specificity for detecting MSI in an East Asian population.
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Affiliation(s)
- Jianmin Zheng
- Department of Pathology, Changhai Hospital of Shanghai, 168 Changhai Road, Shanghai 200433, PR China
| | - Bangxing Huang
- Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, PR China
| | - Xiu Nie
- Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, PR China
| | - Yan Zhu
- Department of Pathology, Changhai Hospital of Shanghai, 168 Changhai Road, Shanghai 200433, PR China
| | - Ningning Han
- Department of Clinical Medicine, Shanghai Tongshu Biotech Co. Ltd, Shanghai 200120, PR China
| | - Yan Li
- Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, PR China
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4
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Multiplex ligation-dependent probe amplification (MLPA) for simultaneous detection of DNA from sunflower, poppy, flaxseed, sesame and soy allergenic ingredients in commercial food products. Food Control 2017. [DOI: 10.1016/j.foodcont.2016.06.014] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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5
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Rodriguez S, Al-Ghamdi OA, Guthrie PA, Shihab HA, McArdle W, Gaunt T, Alharbi KK, Day IN. Frequency of KLK3 gene deletions in the general population. Ann Clin Biochem 2016; 54:472-480. [PMID: 27555663 DOI: 10.1177/0004563216666999] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Background One of the kallikrein genes ( KLK3) encodes prostate-specific antigen, a key biomarker for prostate cancer. A number of factors, both genetic and non-genetic, determine variation of serum prostate-specific antigen concentrations in the population. We have recently found three KLK3 deletions in individuals with very low prostate-specific antigen concentrations, suggesting a link between abnormally reduced KLK3 expression and deletions of KLK3. Here, we aim to determine the frequency of kallikrein gene 3 deletions in the general population. Methods The frequency of KLK3 deletions in the general population was estimated from the 1958 Birth Cohort sample ( n = 3815) using amplification ratiometry control system. In silico analyses using PennCNV were carried out in the same cohort and in NBS-WTCCC2 in order to provide an independent estimation of the frequency of KLK3 deletions in the general population. Results Amplification ratiometry control system results from the 1958 cohort indicated a frequency of KLK3 deletions of 0.81% (3.98% following a less stringent calling criterion). From in silico analyses, we found that potential deletions harbouring the KLK3 gene occurred at rates of 2.13% (1958 Cohort, n = 2867) and 0.99% (NBS-WTCCC2, n = 2737), respectively. These results are in good agreement with our in vitro experiments. All deletions found were in heterozygosis. Conclusions We conclude that a number of individuals from the general population present KLK3 deletions in heterozygosis. Further studies are required in order to know if interpretation of low serum prostate-specific antigen concentrations in individuals with KLK3 deletions may offer false-negative assurances with consequences for prostate cancer screening, diagnosis and monitoring.
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Affiliation(s)
- Santiago Rodriguez
- 1 MRC Integrative Epidemiology Unit (IEU), School of Social and Community Medicine, University of Bristol, Bristol, UK
| | - Osama A Al-Ghamdi
- 2 Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.,3 School of Social and Community Medicine, University of Bristol, Bristol, UK
| | - Philip Ai Guthrie
- 1 MRC Integrative Epidemiology Unit (IEU), School of Social and Community Medicine, University of Bristol, Bristol, UK
| | - Hashem A Shihab
- 1 MRC Integrative Epidemiology Unit (IEU), School of Social and Community Medicine, University of Bristol, Bristol, UK
| | - Wendy McArdle
- 3 School of Social and Community Medicine, University of Bristol, Bristol, UK
| | - Tom Gaunt
- 1 MRC Integrative Epidemiology Unit (IEU), School of Social and Community Medicine, University of Bristol, Bristol, UK
| | - Khalid K Alharbi
- 2 Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia
| | - Ian Nm Day
- 3 School of Social and Community Medicine, University of Bristol, Bristol, UK
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Abstract
Lynch syndrome, an autosomal dominant inherited disorder, is caused by inactivating mutations involving DNA mismatch repair (MMR) genes. This leads to profound genetic instability, including microsatellite instability (MSI) and increased risk for cancer development, particularly colon and endometrial malignancies. Clinical testing of tumor tissues for the presence of MMR gene deficiency is standard practice in clinical oncology, with immunohistochemistry and PCR-based microsatellite instability analysis used as screening tests to identify potential Lynch syndrome families. The ultimate diagnosis of Lynch syndrome requires documentation of mutation within one of the four MMR genes (MLH1, PMS2, MSH2 and MSH6) or EPCAM, currently achieved by comprehensive sequencing analysis of germline DNA. In this review, the genetic basis of Lynch syndrome, methodologies of MMR deficiency testing, and current diagnostic algorithms in the clinical management of Lynch syndrome, are discussed.
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Affiliation(s)
- Natalia Buza
- a Department of Pathology, School of Medicine , Yale University , New Haven , CT , USA
| | - James Ziai
- b Genentech Inc ., San Francisco , CA , USA
| | - Pei Hui
- a Department of Pathology, School of Medicine , Yale University , New Haven , CT , USA
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7
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A founder MLH1 mutation in Lynch syndrome families from Piedmont, Italy, is associated with an increased risk of pancreatic tumours and diverse immunohistochemical patterns. Fam Cancer 2015; 13:401-13. [PMID: 24802709 DOI: 10.1007/s10689-014-9726-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The MLH1 c.2252_2253delAA mutation was found in 11 unrelated families from a restricted area south-west of Turin among 140 families with mutations in the mismatch repair genes. The mutation is located in the highly conserved C-terminal region, responsible for dimerization with the PMS2 protein. Twenty-five tumour tissues from 61 individuals with the c.2252_2253delAA mutation were tested for microsatellite instability (MSI) and protein expression. We compared the clinical features of these families versus the rest of our cohort and screened for a founder effect. All but one tumours showed the MSI-high mutator phenotype. Normal, focal and lack of MLH1 staining were observed in 16, 36 and 48 % of tumours, respectively. PMS2 expression was always lost. The mutation co-segregated with Lynch syndrome-related cancers in all informative families. All families but one fulfilled Amsterdam criteria, a frequency higher than in other MLH1 mutants. This was even more evident for AC II (72.7 vs. 57.5 %). Moreover, all families had at least one colon cancer diagnosed before 50 years and one case with multiple Lynch syndrome-related tumours. Interestingly, a statistically significant (p = 0.0057) higher frequency of pancreatic tumours was observed compared to families with other MLH1 mutations: 8.2 % of affected individuals versus 1.6 %. Haplotype analysis demonstrated a common ancestral origin of the mutation, which originated about 1,550 years ago. The mutation is currently classified as having an uncertain clinical significance. Clinical features, tissue analysis and co-segregation with disease strongly support the hypothesis that the MLH1 c.2252_2253delAA mutation has a pathogenic effect.
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Abstract
Lynch syndrome, which is now recognized as the most common hereditary colorectal cancer condition, is characterized by the predisposition to a spectrum of cancers, primarily colorectal cancer and endometrial cancer. We chronicle over a century of discoveries that revolutionized the diagnosis and clinical management of Lynch syndrome, beginning in 1895 with Warthin's observations of familial cancer clusters, through the clinical era led by Lynch and the genetic era heralded by the discovery of causative mutations in mismatch repair (MMR) genes, to ongoing challenges.
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Affiliation(s)
- Henry T Lynch
- Department of Preventive Medicine and Public Health, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178, USA
| | - Carrie L Snyder
- Department of Preventive Medicine and Public Health, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178, USA
| | - Trudy G Shaw
- Department of Preventive Medicine and Public Health, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178, USA
| | - Christopher D Heinen
- Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3101, USA
| | - Megan P Hitchins
- Department of Medicine (Oncology), Stanford Cancer Institute, Stanford University, Grant Building S169, 1291 Welch Road, Stanford, California 94305, USA
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9
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Liu M, Hu P, Zhang G, Zeng Y, Yang H, Fan J, Jin L, Liu H, Deng Y, Li S, Zeng X, Elingarami S, He N. Copy number variation analysis by ligation-dependent PCR based on magnetic nanoparticles and chemiluminescence. Am J Cancer Res 2015; 5:71-85. [PMID: 25553099 PMCID: PMC4265749 DOI: 10.7150/thno.10117] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2014] [Accepted: 09/21/2014] [Indexed: 12/19/2022] Open
Abstract
A novel system for copy number variation (CNV) analysis was developed in the present study using a combination of magnetic separation and chemiluminescence (CL) detection technique. The amino-modified probes were firstly immobilized onto carboxylated magnetic nanoparticles (MNPs) and then hybridized with biotin-dUTP products, followed by amplification with ligation-dependent polymerase chain reaction (PCR). After streptavidin-modified alkaline phosphatase (STV-AP) bonding and magnetic separation, the CL signals were then detected. Results showed that the quantification of PCR products could be reflected by CL signal values. Under optimum conditions, the CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with logarithm of the target concentration. To validate the methodology, copy numbers of six genes from the human genome were detected. To compare the detection accuracy, multiplex ligation-dependent probe amplification (MLPA) and MNPs-CL detection were performed. Overall, there were two discrepancies by MLPA analysis, while only one by MNPs-CL detection. This research demonstrated that the novel MNPs-CL system is a useful analytical tool which shows simple, sensitive, and specific characters which are suitable for CNV analysis. Moreover, this system should be improved further and its application in the genome variation detection of various diseases is currently under further investigation.
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10
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Bashyam MD, Kotapalli V, Raman R, Chaudhary AK, Yadav BK, Gowrishankar S, Uppin SG, Kongara R, Sastry RA, Vamsy M, Patnaik S, Rao S, Dsouza S, Desai D, Tester A. Evidence for presence of mismatch repair gene expression positive Lynch syndrome cases in India. Mol Carcinog 2014; 54:1807-14. [PMID: 25420488 DOI: 10.1002/mc.22244] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2014] [Accepted: 10/01/2014] [Indexed: 01/09/2023]
Abstract
Lynch syndrome (LS), the most common form of familial CRC predisposition that causes tumor onset at a young age, is characterized by the presence of microsatellite instability (MSI) in tumors due to germline inactivation of mismatch repair (MMR) system. Two MMR genes namely MLH1 and MSH2 account for majority of LS cases while MSH6 and PMS2 may account for a minor proportion. In order to identify MMR genes causing LS in India, we analyzed MSI and determined expression status of the four MMR genes in forty eight suspected LS patient colorectal tumor samples. Though a majority exhibited MSI, only 58% exhibited loss of MMR expression, a significantly low proportion compared to reports from other populations. PCR-DNA sequencing and MLPA-based mutation and exonic deletion/duplication screening respectively, revealed genetic lesions in samples with and without MMR gene expression. Interestingly, tumor samples with and without MMR expression exhibited significant differences with respect to histological (mucin content) and molecular (instability exhibited by mononucleotide microsatellites) features. The study has revealed for the first time a significant proportion of LS tumors not exhibiting loss of MMR expression.
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Affiliation(s)
- Murali D Bashyam
- Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India
| | - Viswakalyan Kotapalli
- Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India
| | - Ratheesh Raman
- Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India
| | - Ajay K Chaudhary
- Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India
| | - Brijesh K Yadav
- Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India
| | | | | | | | | | - Mohana Vamsy
- Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, India
| | - Sujit Patnaik
- Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, India
| | - Satish Rao
- Krishna Institute of Medical Sciences, Hyderabad, India
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Mancini-DiNardo D, Judkins T, Woolstenhulme N, Burton C, Schoenberger J, Ryder M, Murray A, Gutin N, Theisen A, Holladay J, Craft J, Arnell C, Moyes K, Roa B. Design and validation of an oligonucleotide microarray for the detection of genomic rearrangements associated with common hereditary cancer syndromes. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2014; 33:74. [PMID: 25204323 PMCID: PMC4174268 DOI: 10.1186/s13046-014-0074-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/22/2014] [Accepted: 08/29/2014] [Indexed: 12/14/2022]
Abstract
Background Conventional Sanger sequencing reliably detects the majority of genetic mutations associated with hereditary cancers, such as single-base changes and small insertions or deletions. However, detection of genomic rearrangements, such as large deletions and duplications, requires special technologies. Microarray analysis has been successfully used to detect large rearrangements (LRs) in genetic disorders. Methods We designed and validated a high-density oligonucleotide microarray for the detection of gene-level genomic rearrangements associated with hereditary breast and ovarian cancer (HBOC), Lynch, and polyposis syndromes. The microarray consisted of probes corresponding to the exons and flanking introns of BRCA1 and BRCA2 (≈1,700) and Lynch syndrome/polyposis genes MLH1, MSH2, MSH6, APC, MUTYH, and EPCAM (≈2,200). We validated the microarray with 990 samples previously tested for LR status in BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, MUTYH, or EPCAM. Microarray results were 100% concordant with previous results in the validation studies. Subsequently, clinical microarray analysis was performed on samples from patients with a high likelihood of HBOC mutations (13,124), Lynch syndrome mutations (18,498), and polyposis syndrome mutations (2,739) to determine the proportion of LRs. Results Our results demonstrate that LRs constitute a substantial proportion of genetic mutations found in patients referred for hereditary cancer genetic testing. Conclusion The use of microarray comparative genomic hybridization (CGH) for the detection of LRs is well-suited as an adjunct technology for both single syndrome (by Sanger sequencing analysis) and extended gene panel testing by next generation sequencing analysis. Genetic testing strategies using microarray analysis will help identify additional patients carrying LRs, who are predisposed to various hereditary cancers.
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12
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Wielders EAL, Hettinger J, Dekker R, Kets CM, Ligtenberg MJ, Mensenkamp AR, van den Ouweland AMW, Prins J, Wagner A, Dinjens WNM, Dubbink HJ, van Hest LP, Menko F, Hogervorst F, Verhoef S, te Riele H. Functional analysis of MSH2 unclassified variants found in suspected Lynch syndrome patients reveals pathogenicity due to attenuated mismatch repair. J Med Genet 2014; 51:245-53. [PMID: 24501230 DOI: 10.1136/jmedgenet-2013-101987] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND Lynch syndrome, an autosomal-dominant disorder characterised by high colorectal and endometrial cancer risks, is caused by inherited mutations in DNA mismatch repair (MMR) genes. Mutations fully abrogating gene function are unambiguously disease causing. However, missense mutations often have unknown functional implications, hampering genetic counselling. We have applied a novel approach to study three MSH2 unclassified variants (UVs) found in Dutch families with suspected Lynch syndrome. METHODS The three mutations were recreated in the endogenous Msh2 gene in mouse embryonic stem cells by oligonucleotide-directed gene modification. The effect of the UVs on MMR activity was then tested using a set of functional assays interrogating the main MMR functions. RESULTS We recreated and functionally tested three MSH2 UVs: MSH2-Y165D (c.493T>G), MSH2-Q690E (c.2068C>G) and MSH2-M813V (c.2437A>G). We observed reduced levels of MSH2-Y165D and MSH2-Q690E but not MSH2-M813V proteins. MSH2-M813V was able to support all MMR functions similar to wild-type MSH2, whereas MSH2-Y165D and MSH2-Q690E showed partial defects. CONCLUSIONS Based on the results from our functional assays, we conclude that the MSH2-M813V variant is not disease causing. The MSH2-Y165D and MSH2-Q690E variants affect MMR function and are therefore likely the underlying cause of familial cancer predisposition. Since the MMR defect is partial, these variants may represent low penetrance alleles.
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Affiliation(s)
- Eva A L Wielders
- Division of Biological Stress Response, The Netherlands Cancer Institute, Amsterdam, The Netherlands
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Majewski IJ, Kluijt I, Cats A, Scerri TS, de Jong D, Kluin RJC, Hansford S, Hogervorst FBL, Bosma AJ, Hofland I, Winter M, Huntsman D, Jonkers J, Bahlo M, Bernards R. An α-E-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer. J Pathol 2013. [PMID: 23208944 DOI: 10.1002/path.4152] [Citation(s) in RCA: 158] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Diffuse gastric cancers typically present as late-stage tumours and, as a result, the 5 year survival rate is poor. Some gastric cancers are hereditary and these tend to be of the diffuse type; 30-40% of hereditary diffuse gastric cancers (HDGCs) can be explained by defective germline alleles of E-cadherin (CDH1), but for the remaining families the factors driving susceptibility remain unknown. We had access to a large HDGC pedigree with no obvious mutation in CDH1, and applied exome sequencing to identify new genes involved in gastric cancer. We identified a germline truncating allele of α-E-catenin (CTNNA1) that was present in two family members with invasive diffuse gastric cancer and four in which intramucosal signet ring cells were detected as part of endoscopic surveillance. The remaining CTNNA1 allele was silenced in the two diffuse gastric cancers from the family that were available for screening, and this was also true for signet ring cells identified in endoscopic biopsies. Since α-E-catenin functions in the same complex as E-cadherin, our results call attention to the broader signalling network surrounding these proteins in HDGC. We also detected somatic mutations in one tumour and found substantial overlap with genes mutated in sporadic gastric cancer, including PIK3CA, ARID1A, MED12 and MED23.
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Affiliation(s)
- Ian J Majewski
- Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands
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Gullapalli RR, Desai KV, Santana-Santos L, Kant JA, Becich MJ. Next generation sequencing in clinical medicine: Challenges and lessons for pathology and biomedical informatics. J Pathol Inform 2012; 3:40. [PMID: 23248761 PMCID: PMC3519097 DOI: 10.4103/2153-3539.103013] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2012] [Accepted: 07/19/2012] [Indexed: 11/25/2022] Open
Abstract
The Human Genome Project (HGP) provided the initial draft of mankind's DNA sequence in 2001. The HGP was produced by 23 collaborating laboratories using Sanger sequencing of mapped regions as well as shotgun sequencing techniques in a process that occupied 13 years at a cost of ~$3 billion. Today, Next Generation Sequencing (NGS) techniques represent the next phase in the evolution of DNA sequencing technology at dramatically reduced cost compared to traditional Sanger sequencing. A single laboratory today can sequence the entire human genome in a few days for a few thousand dollars in reagents and staff time. Routine whole exome or even whole genome sequencing of clinical patients is well within the realm of affordability for many academic institutions across the country. This paper reviews current sequencing technology methods and upcoming advancements in sequencing technology as well as challenges associated with data generation, data manipulation and data storage. Implementation of routine NGS data in cancer genomics is discussed along with potential pitfalls in the interpretation of the NGS data. The overarching importance of bioinformatics in the clinical implementation of NGS is emphasized.[7] We also review the issue of physician education which also is an important consideration for the successful implementation of NGS in the clinical workplace. NGS technologies represent a golden opportunity for the next generation of pathologists to be at the leading edge of the personalized medicine approaches coming our way. Often under-emphasized issues of data access and control as well as potential ethical implications of whole genome NGS sequencing are also discussed. Despite some challenges, it's hard not to be optimistic about the future of personalized genome sequencing and its potential impact on patient care and the advancement of knowledge of human biology and disease in the near future.
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Affiliation(s)
- Rama R Gullapalli
- Department of Pathology, University of Pittsburgh Medical Centre, A701, Scaife Hall, 3550 Terrace Street, Pittsburgh, PA
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Borelli I, Barberis MA, Spina F, Casalis Cavalchini GC, Vivanet C, Balestrino L, Micheletti M, Allavena A, Sala P, Carcassi C, Pasini B. A unique MSH2 exon 8 deletion accounts for a major portion of all mismatch repair gene mutations in Lynch syndrome families of Sardinian origin. Eur J Hum Genet 2012; 21:154-61. [PMID: 22781090 DOI: 10.1038/ejhg.2012.150] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Lynch syndrome is an autosomal-dominant hereditary condition predisposing to the development of specific cancers, because of germline mutations in the DNA-mismatch repair (MMR) genes. Large genomic deletions represent a significant fraction of germline mutations, particularly among the MSH2 gene, in which they account for 20% of the mutational spectrum. In this study we analyzed 13 Italian families carrying MSH2 exon 8 deletions, 10 of which of ascertained Sardinian origin. The overrepresentation of Sardinians was unexpected, as families from Sardinia account for a small quota of MMR genes mutation tests performed in our laboratory. The hypothesis that such a result is owing to founder effects in Sardinia was tested by breakpoint junctions sequencing and haplotype analyses. Overall, five different exon eight deletions were identified, two of which recurrent in families, all apparently unrelated, of Sardinian origin (one in eight families, one in two families). The c.1277-1180_1386+2226del3516insCATTCTCTTTGAAAA deletion shares the same haplotype between all families and appears so far restricted to the population of South-West Sardinia, showing the typical features of a founder effect. The three non-Sardinian families showed three different breakpoint junctions and haplotypes, suggesting independent mutational events. This work has useful implications in genetic testing for Lynch syndrome. We developed a quick test for each of the identified deletions: this can be particularly useful in families of Sardinian origin, in which MSH2 exon 8 deletions may represent 50% of the overall mutational spectrum of the four MMR genes causing Lynch syndrome.
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Affiliation(s)
- Iolanda Borelli
- Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 19, Turin, Italy.
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Medeiros F, Lindor NM, Couch FJ, Highsmith WE. The germline MLH1 K618A variant and susceptibility to Lynch syndrome-associated tumors. J Mol Diagn 2012; 14:264-73. [PMID: 22426235 DOI: 10.1016/j.jmoldx.2012.01.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2011] [Revised: 12/22/2011] [Accepted: 01/10/2012] [Indexed: 12/31/2022] Open
Abstract
Missense variants discovered during sequencing of cancer susceptibility genes can be problematic for clinical interpretation. MLH1 K618A, which results from a 2-bp alteration (AAG→GCG) leading to a substitution of lysine to alanine in codon 618, has variously been interpreted as a pathogenic mutation, a variant of unknown significance, and a benign polymorphism. We evaluated the role of MLH1 K618A in predisposition to cancer by genotyping 1512 control subjects to assess its frequency in the general population. We also reviewed the literature concerning MLH1 K618A in families with colorectal cancer. The measured allele frequency of the K618A variant was 0.40%, which is remarkably close to the 0.44% summarized from 2491 control subjects in the literature. K618A was over-represented in families with suspected Lynch syndrome. In 1366 families, the allele frequency was 0.88% (OR = 2.1, 95% CI = 1.3 to 3.5; P = 0.006). In studies of sporadic cancers of the type associated with Lynch syndrome, K618A was over-represented in 1742 cases (allele frequency of 0.83) (OR = 2.0, 95% CI = 1.2 to 3.2; P = 0.008). We conclude that MLH1 K618A is not a fully penetrant Lynch syndrome mutation, although it is not without effect, appearing to increase the risk of Lynch syndrome-associated tumors approximately twofold. Our systematic assessment approach may be useful for variants in other genes.
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Affiliation(s)
- Fabiola Medeiros
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
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Ehlert A, Demmel A, Hupfer C, Busch U, Engel KH. Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2011; 26:409-18. [PMID: 19680915 DOI: 10.1080/02652030802593529] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg(-1) range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg(-1). The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.
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Affiliation(s)
- Alexandra Ehlert
- Department of General Food Technology, Technische Universität München, Freising-Weihenstephan D-85350, Germany
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A novel exonic rearrangement affecting MLH1 and the contiguous LRRFIP2 is a founder mutation in Portuguese Lynch syndrome families. Genet Med 2011; 13:895-902. [DOI: 10.1097/gim.0b013e31821dd525] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
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Dahdaleh FS, Carr JC, Calva D, Howe JR. Juvenile polyposis and other intestinal polyposis syndromes with microdeletions of chromosome 10q22-23. Clin Genet 2011; 81:110-6. [PMID: 21834858 DOI: 10.1111/j.1399-0004.2011.01763.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Juvenile polyposis (JP) is an autosomal dominant hamartomatous polyposis syndrome that carries a significant risk for the development of colorectal cancer. Microdeletions of one of the two predisposing genes to JP, BMPR1A, have been associated with a severe form of JP called juvenile polyposis of infancy. Many of these deletions have also been found to contiguously include PTEN, which is the gene responsible for the development of Cowden syndrome. The advent of molecular techniques that localize genomic copy number variations and others that target specific genes such as multiplex-ligation probe analysis has allowed researchers to explore this area further for deletions. Here, we review the literature for microdeletions described on chromosome 10q22-23 in patients with JP and other intestinal polyposis syndromes.
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Affiliation(s)
- F S Dahdaleh
- Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, IA, USA
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Abstract
Colorectal cancer is common in the Western world; ~5% of individuals diagnosed with colorectal cancer have an identifiable inherited genetic predisposition to this malignancy. Genetic testing and rational clinical management recommendations currently exist for the management of individuals with a variety of colorectal cancer syndromes, including hereditary nonpolyposis colorectal cancer (HNPCC, also known as Lynch syndrome), familial adenomatous polyposis (FAP), MYH-associated polyposis (MAP), and the hamartomatous polyposis syndromes (Peutz-Jeghers, juvenile polyposis, and Cowden disease). In addition to colorectal neoplasia, these syndromes frequently predispose carriers to a variety of extracolonic cancers. The elucidation of the genetic basis of several colorectal cancer predisposition syndromes over the past two decades has allowed for better management of individuals who are either affected with, or at-risk for inherited colorectal cancer syndromes. Appropriate multidisciplinary management of these individuals includes genetic counseling, genetic testing, clinical screening, and treatment recommendations.
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Affiliation(s)
- Robert Gryfe
- Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada
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Abstract
PURPOSE Lynch syndrome accounts for 2-4% of all colorectal cancer, and is mainly caused by germline mutations in the DNA mismatch repair genes. Our aim was to characterize the genetic mutation responsible for Lynch syndrome in an extensive Colombian family and to study its prevalence in Antioquia. METHODS A Lynch syndrome family fulfilling Amsterdam criteria II was studied by immunohistochemistry and by multiplex ligation-dependent probe amplification (MLPA). Results were confirmed by additional independent MLPA, Southern blotting, and sequencing. RESULTS Index case tumor immunohistochemistry results were MLH1-, MSH2+, MSH6+, and PMS2-. MLPA analysis detected a duplication of exons 12 and 13 of MLH1. This mutation was confirmed and characterized precisely to span 4219 base pairs. Duplication screening in this family led to the identification of six additional carriers and 13 noncarriers. We also screened 123 early-onset independent colorectal cancer cases from the same area and identified an additional unrelated carrier. CONCLUSION A novel duplication of exons 12 and 13 of the MLH1 gene was detected in two independent Lynch syndrome families from Colombia. A putative founder effect and prescreening Lynch syndrome Antioquia families for this specific mutation before thorough mismatch repair mutational screening could be suggested.
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Herkert JC, Niessen RC, Olderode-Berends MJW, Veenstra-Knol HE, Vos YJ, van der Klift HM, Scheenstra R, Tops CMJ, Karrenbeld A, Peters FTM, Hofstra RMW, Kleibeuker JH, Sijmons RH. Paediatric intestinal cancer and polyposis due to bi-allelic PMS2 mutations: case series, review and follow-up guidelines. Eur J Cancer 2011; 47:965-82. [PMID: 21376568 DOI: 10.1016/j.ejca.2011.01.013] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2010] [Accepted: 01/20/2011] [Indexed: 11/30/2022]
Abstract
BACKGROUND Bi-allelic germline mutations of one of the DNA mismatch repair genes, so far predominantly found in PMS2, cause constitutional MMR-deficiency syndrome. This rare disorder is characterised by paediatric intestinal cancer and other malignancies. We report the clinical, immunohistochemical and genetic characterisation of four families with bi-allelic germline PMS2 mutations. We present an overview of the published gastrointestinal manifestations of CMMR-D syndrome and propose recommendations for gastro-intestinal screening. METHODS AND RESULTS The first proband developed a cerebral angiosarcoma at age 2 and two colorectal adenomas at age 7. Genetic testing identified a complete PMS2 gene deletion and a frameshift c.736_741delinsTGTGTGTGAAG (p.Pro246CysfsX3) mutation. In the second family, both the proband and her brother had multiple intestinal adenomas, initially wrongly diagnosed as familial adenomatous polyposis. A splice site c.2174+1G>A, and a missense c.137G>T (p.Ser46Ile) mutation in PMS2 were identified. The third patient was diagnosed with multiple colorectal adenomas at age 11; he developed a high-grade dysplastic colorectal adenocarcinoma at age 21. Two intragenic PMS2 deletions were found. The fourth proband developed a cerebral anaplastic ganglioma at age 9 and a high-grade colerectal dysplastic adenoma at age 10 and carries a homozygous c.2174+1G>A mutation. Tumours of all patients showed microsatellite instability and/or loss of PMS2 expression. CONCLUSIONS Our findings show the association between bi-allelic germline PMS2 mutations and severe childhood-onset gastrointestinal manifestations, and support the notion that patients with early-onset gastrointestinal adenomas and cancer should be investigated for CMMR-D syndrome. We recommend yearly follow-up with colonoscopy from age 6 and simultaneous video-capsule small bowel enteroscopy from age 8.
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Affiliation(s)
- Johanna C Herkert
- Department of Genetics, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.
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Houdayer C, Dehainault C, Gauthier-Villars M, Stoppa-Lyonnet D. Screening for genomic rearrangements by multiplex PCR/liquid chromatography. Methods Mol Biol 2011; 688:127-42. [PMID: 20938836 DOI: 10.1007/978-1-60761-947-5_9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Screening for large gene rearrangements has become established as an important part of molecular medicine; however, it is also challenging as these rearrangements range from an extra copy of a complete chromosome(s) to deletion or duplication of a single exon. In this chapter, we describe a versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography (MP/LC) assay. Multiple genomic fragments are amplified under semiquantitative conditions using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography, and quantitated by fluorescent detection using a postcolumn intercalation dye. The relative peak intensities for each target directly reflect DNA copy number. This technique can be used not only to screen intronic, exonic, and intergenic parts of the genome but also for transcript quantitation. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to DHPLC users, as it broadens the spectrum of available applications on a DHPLC system. The authors describe a detailed protocol for large rearrangement screening in the RB1 gene.
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Affiliation(s)
- Claude Houdayer
- Service de Génétique Oncologique, Institut Curie Hôpital, Paris, France.
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Hermsen M, Coffa J, Ylstra B, Meijer G, Morreau H, van Eijk R, Oosting J, van Wezel T. High‐Resolution Analysis of Genomic Copy Number Changes. Genomics 2010. [DOI: 10.1002/9780470711675.ch1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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van Riel E, Ausems MG, Hogervorst FB, Kluijt I, van Gijn ME, van Echtelt J, Scheidel-Jacobse K, Hennekam EF, Stulp RP, Vos YJ, Offerhaus GJA, Menko FH, Gille JJ. A novel pathogenic MLH1 missense mutation, c.112A > C, p.Asn38His, in six families with Lynch syndrome. Hered Cancer Clin Pract 2010; 8:7. [PMID: 20704743 PMCID: PMC2927519 DOI: 10.1186/1897-4287-8-7] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2010] [Accepted: 08/12/2010] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND An unclassified variant (UV) in exon 1 of the MLH1 gene, c.112A > C, p.Asn38His, was found in six families who meet diagnostic criteria for Lynch syndrome. The pathogenicity of this variant was unknown. We aim to elucidate the pathogenicity of this MLH1 variant in order to counsel these families adequately and to enable predictive testing in healthy at-risk relatives. METHODS We studied clinical data, microsatellite instability and immunohistochemical staining of MMR proteins, and performed genealogy, haplotype analysis and DNA testing of control samples. RESULTS The UV showed co-segregation with the disease in all families. All investigated tumors showed a microsatellite instable pattern. Immunohistochemical data were variable among tested tumors. Three families had a common ancestor and all families originated from the same geographical area in The Netherlands. Haplotype analysis showed a common haplotype in all six families. CONCLUSIONS We conclude that the MLH1 variant is a pathogenic mutation and genealogy and haplotype analysis results strongly suggest that it is a Dutch founder mutation. Our findings imply that predictive testing can be offered to healthy family members. The immunohistochemical data of MMR protein expression show that interpreting these results in case of a missense mutation should be done with caution.
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Affiliation(s)
- Els van Riel
- Department of Medical Genetics, University Medical Centre Utrecht, Lundlaan 6, Utrecht, The Netherlands.
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Abstract
Lynch syndrome (LS), or hereditary nonpolyposis colorectal cancer, is the most common hereditary colorectal cancer (CRC) syndrome, accounting for approximately 2-5% of all newly diagnosed cases of CRC. Patients with LS have an increased lifetime risk of colorectal (52.2% in women and 68.7% in men) and endometrial cancer (15-70%), as well as certain extra-colonic cancers. Germline mutations in one of several DNA mismatch repair genes underlie LS. Molecular testing has emerged as an indispensable strategy for the diagnosis of LS. The diagnostic work-up of at-risk individuals includes a careful family history evaluation, microsatellite instability, immunohistochemistry and germline DNA analysis. A positive test result can guide clinicians in formulating the appropriate screening, surveillance and management strategies. However, because of the absence of an overt phenotype, such as a diffuse polyposis, it is not always straightforward to recognize LS clinically.
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Affiliation(s)
- Maria S Pino
- Gastrointestinal Unit, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA
| | - Daniel C Chung
- Gastrointestinal Unit, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA
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A study on MSH2 and MLH1 mutations in hereditary nonpolyposis colorectal cancer families from the Basque Country, describing four new germline mutations. Fam Cancer 2010; 8:533-9. [PMID: 19760518 DOI: 10.1007/s10689-009-9283-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome underlies between 2 and 5% of all colorectal cancer. It is inherited as an autosomal dominant condition due to mutations in the mismatch repair genes. Fifty-four non-related index cases, 21 of them fulfilling Amsterdam criteria I or II, were studied. Ten (10/21 = 47.6%) different pathological mutations were found in this group, two of which had not previously been reported--one in MLH1 and the other in MSH2-. In the remaining patients, we also found another family with one of these new mutations, and four additional changes, two of which were also new--a pathological change in MSH2 and a second change of uncertain significance in MLH1-, while the other two changes had already been reported. Of all mutations, eight were found in MSH2 (8/15 = 53.3%) and seven in MLH1 (7/15 = 46.6%), suggesting a slightly greater involvement of MSH2 in HNPCC than MLH1 in our population, in contrast to the results reported by other authors.
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Zavodna K, Krivulcik T, Bujalkova MG, Slamka T, Martinicky D, Ilencikova D, Bartosova Z. Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers. BMC Cancer 2009; 9:405. [PMID: 19930554 PMCID: PMC2788582 DOI: 10.1186/1471-2407-9-405] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2009] [Accepted: 11/20/2009] [Indexed: 11/24/2022] Open
Abstract
Background Depending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts. Methods The main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes. Results We found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region. Conclusion LGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.
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Affiliation(s)
- Katarina Zavodna
- Laboratory of Cancer Genetics, Cancer Research Institute of Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic.
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Abstract
Lynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome attributable to deleterious germline mutations in mismatch repair (MMR) genes. The syndrome is typified by early-onset, frequently right-sided colorectal cancers (CRCs) with characteristic histologic features and tendency for multiplicity and an increased risk for extracolonic tumors at particular sites; it accounts for 1% to 5% of CRC. Deficient mismatch repair (dMMR) function manifests as immunohistochemically detectable absence of one or more MMR proteins and microsatellite instability (MSI). Approximately 15% of sporadic, noninherited CRC are characterized by high-level MSI, nearly always owing to transcriptional silencing of MLH1; these sporadic and LS cases exhibit considerable phenotypic overlap. Identification of CRC with dMMR is desirable to identify LS and because MSI status is prognostic and potentially predictive. This review will discuss the history of LS, the principles of MMR and MSI, the clinicopathologic features of LS-associated and sporadic high-level MSI CRC, the fundamentals of clinical testing for dMMR CRC, and the results of the Columbus-area Lynch syndrome study. We conclude with our approach to population-based LS screening based on institutional experience with nearly 2000 cases.
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Niessen RC, Hofstra RMW, Westers H, Ligtenberg MJL, Kooi K, Jager POJ, de Groote ML, Dijkhuizen T, Olderode-Berends MJW, Hollema H, Kleibeuker JH, Sijmons RH. Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer 2009; 48:737-44. [PMID: 19455606 DOI: 10.1002/gcc.20678] [Citation(s) in RCA: 145] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
It was shown that Lynch syndrome can be caused by germline hypermethylation of the MLH1 and MSH2 promoters. Furthermore, it has been demonstrated very recently that germline deletions of the 3' region of EPCAM cause transcriptional read-through which results in silencing of MSH2 by hypermethylation. We wanted to determine the prevalence of germline MLH1 promoter hypermethylation and of germline and somatic MSH2 promoter hypermethylation in a large group of Lynch syndrome-suspected patients. From a group of 331 Lynch Syndrome-suspected patients we selected cases, who had no germline MLH1, MSH2, or MSH6 mutation and whose tumors showed loss of MLH1 or MSH2, or, if staining was unavailable, had a tumor with microsatellite instability. Methylation assays were performed to test these patients for germline MLH1 and/or MSH2 promoter hypermethylation. Two patients with germline MLH1 promoter hypermethylation and no patients with germline MSH2 promoter hypermethylation were identified. In the subgroup screened for germline MSH2 promoter hypermethylation, we identified 3 patients with somatic MSH2 promoter hypermethylation in their tumors, which was caused by a germline EPCAM deletion. In the group of 331 Lynch Syndrome-suspected patients, the frequencies of germline MLH1 promoter hypermethylation and somatic MSH2 promoter hypermethylation caused by germline EPCAM deletions are 0.6 and 0.9%, respectively. These mutations, therefore, seem to be rather infrequent. However, the contribution of germline MLH1 hypermethylation and EPCAM deletions to the genetically proven Lynch syndrome cases in this cohort is very high. Previously 27 pathogenic mutations were identified; the newly identified mutations now represent 16% of all mutations.
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Affiliation(s)
- Renée C Niessen
- Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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SUTHERLAND MS, KEENEY S, BOLTON-MAGGS PHB, HAY CRM, WILL A, CUMMING AM. The mutation spectrum associated with type 3 von Willebrand disease in a cohort of patients from the North West of England. Haemophilia 2009; 15:1048-57. [DOI: 10.1111/j.1365-2516.2009.02059.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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Gylling A, Ridanpää M, Vierimaa O, Aittomäki K, Avela K, Kääriäinen H, Laivuori H, Pöyhönen M, Sallinen SL, Wallgren-Pettersson C, Järvinen HJ, Mecklin JP, Peltomäki P. Large genomic rearrangements and germline epimutations in Lynch syndrome. Int J Cancer 2009; 124:2333-40. [PMID: 19173287 DOI: 10.1002/ijc.24230] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
In one-third of families fulfilling the Amsterdam criteria for hereditary nonpolyposis colorectal cancer/Lynch syndrome, and a majority of those not fulfilling these criteria point mutations in DNA mismatch repair (MMR) genes are not found. The role of large genomic rearrangements and germline epimutations in MLH1, MSH2 and MSH6 was evaluated in 2 such cohorts. All 45 index patients were mutation-negative by genomic sequencing and testing for a prevalent population-specific founder mutation, and selectively lacked MMR protein expression in tumor tissue. Eleven patients ("research cohort") represented 11 mutation-negative families among 81 verified or putative Lynch syndrome families from the nation-wide Hereditary Colorectal Cancer Registry of Finland. Thirty-four patients from 33 families ("clinic-based cohort") represented suspected Lynch syndrome patients tested for MMR gene mutations in a diagnostic laboratory during 2004-2007. Multiplex ligation-dependent probe amplification (MLPA) and methylation-specific (MS)-MLPA were used to detect rearrangements and epimutations, respectively. Large genomic deletions occurred in 12/45 patients (27%), being present in 3/25 (12%), 9/16 (56%) and 0/4 (0%) among index patients lacking MLH1, MSH2 or MSH6 expression, respectively. Germline epimutations of MLH1, one of which coexisted with a genomic deletion, occurred in 2 patients (4%) and were accompanied by monoallelic expression in mRNA. Large genomic deletions (mainly MSH2) and germline epimutations (MLH1) together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of such families.
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Affiliation(s)
- Annette Gylling
- Department of Medical Genetics, University of Helsinki, Helsinki, Finland.
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Sheng JQ, Zhang H, Ji M, Fu L, Mu H, Zhang MZ, Huang JS, Han M, Li AQ, Wei Z, Sun ZQ, Wu ZT, Xia CH, Li SR. Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer. World J Gastroenterol 2009; 15:983-9. [PMID: 19248199 PMCID: PMC2653409 DOI: 10.3748/wjg.15.983] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods.
METHODS: A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability (MSI), gene mutation and methylation detection.
RESULTS: High frequency microsatellite instability occurred in 25 probands (83.3%) of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic mutation occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%.
CONCLUSION: Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.
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da Silva FCC, Valentin MD, Ferreira FDO, Carraro DM, Rossi BM. Mismatch repair genes in Lynch syndrome: a review. SAO PAULO MED J 2009; 127:46-51. [PMID: 19466295 PMCID: PMC10969316 DOI: 10.1590/s1516-31802009000100010] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2008] [Revised: 12/07/2008] [Accepted: 12/09/2008] [Indexed: 01/01/2023] Open
Abstract
Lynch syndrome represents 1-7% of all cases of colorectal cancer and is an autosomal-dominant inherited cancer predisposition syndrome caused by germline mutations in deoxyribonucleic acid (DNA) mismatch repair genes. Since the discovery of the major human genes with DNA mismatch repair function, mutations in five of them have been correlated with susceptibility to Lynch syndrome: mutS homolog 2 (MSH2); mutL homolog 1 (MLH1); mutS homolog 6 (MSH6); postmeiotic segregation increased 2 (PMS2); and postmeiotic segregation increased 1 (PMS1). It has been proposed that one additional mismatch repair gene, mutL homolog 3 (MLH3), also plays a role in Lynch syndrome predisposition, but the clinical significance of mutations in this gene is less clear. According to the InSiGHT database (International Society for Gastrointestinal Hereditary Tumors), approximately 500 different LS-associated mismatch repair gene mutations are known, primarily involving MLH1 (50%) and MSH2 (40%), while others account for 10%. Much progress has been made in understanding the molecular basis of Lynch Syndrome. Molecular characterization will be the most accurate way of defining Lynch syndrome and will provide predictive information of greater accuracy regarding the risks of colon and extracolonic cancer and enable optimal cancer surveillance regimens.
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Affiliation(s)
| | | | - Fábio de Oliveira Ferreira
- MD, PhD. Surgeon at Hospital AC Camargo and researcher in the Research Center of Hospital AC Camargo, São Paulo, Brazil.
| | - Dirce Maria Carraro
- PhD. Researcher in the Research Center of Hospital AC Camargo, São Paulo, Brazil.
| | - Benedito Mauro Rossi
- MD, PhD. Surgeon at Hospital AC Camargo and researcher in the Research Center of Hospital AC Camargo, São Paulo, Brazil.
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Vignoli M, Scaini MC, Ghiorzo P, Sestini R, Bruno W, Menin C, Gensini F, Piazzini M, Testori A, Manoukian S, Orlando C, D'Andrea E, Bianchi-Scarrà G, Genuardi M. Genomic rearrangements of the CDKN2A locus are infrequent in Italian malignant melanoma families without evidence of CDKN2A/CDK4 point mutations. Melanoma Res 2008; 18:431-437. [PMID: 19011513 DOI: 10.1097/cmr.0b013e328319412f] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Predisposition to familial cutaneous malignant melanoma has been associated with mutations in the CDKN2A and CDK4 genes. However, only a small subgroup of melanoma pedigrees harbour CDKN2A or CDK4 germline mutations. It is possible that other types of CDKN2A rearrangements, not detectable by routine PCR-based approaches, are involved in a fraction of melanoma cases negative for point sequence changes. In order to gain insights on the possible role of CDKN2A large deletions or duplications in melanoma susceptibility in the Italian population, we screened a series of 124 cutaneous malignant melanoma families referred to five national medical/cancer genetics centres. All probands were negative for point mutations in CDKN2A and CDK4. All samples were tested by MLPA (multiplex ligation-dependent probe amplification), and the results were confirmed by real-time quantitative PCR in a subset of 53 cases. No genomic rearrangements were detected in this series, one of the largest so far investigated. These data suggest that large deletions/duplications in the CDKN2A locus are infrequently involved in the development of familial melanoma in the Italian population. Based on these results, routine search for these rearrangements in CDKN2A- and CDK4-mutation negative melanoma families is not warranted, although it would be reasonable to pursue it in selected cases with very strong family history and/or showing linkage to 9p21.
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Affiliation(s)
- Marina Vignoli
- Fiorgen Foundation for Pharmacogenomics, Sesto Fiorentino, Italy
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Wang JW, Kurahashi H, Ishii A, Kojima T, Ohfu M, Inoue T, Ogawa A, Yasumoto S, Oguni H, Kure S, Fujii T, Ito M, Okuno T, Shirasaka Y, Natsume J, Hasegawa A, Konagaya A, Kaneko S, Hirose S. Microchromosomal deletions involvingSCN1Aand adjacent genes in severe myoclonic epilepsy in infancy. Epilepsia 2008; 49:1528-34. [DOI: 10.1111/j.1528-1167.2008.01609.x] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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KEENEY S, BOWEN D, CUMMING A, ENAYAT S, GOODEVE A, HILL M. The molecular analysis of von Willebrand disease: a guideline from the UK Haemophilia Centre Doctors’ Organisation Haemophilia Genetics Laboratory Network. Haemophilia 2008; 14:1099-111. [DOI: 10.1111/j.1365-2516.2008.01813.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Gomez LC, Marzese DM, Adi J, Bertani D, Ibarra J, Mol B, Vos IJ, De Marchi G, Roqué M. MLPA mutation detection in Argentine HNPCC and FAP families. Fam Cancer 2008; 8:67-73. [PMID: 18615272 DOI: 10.1007/s10689-008-9200-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2008] [Accepted: 06/11/2008] [Indexed: 11/29/2022]
Abstract
Colorectal cancer (CC) is the secondary cause of death in the Western countries of which approximately 15% are considered to be hereditary. The hereditary forms are Familial Adenomatous Polyposis (FAP) and Hereditary Non Polyposis Colorectal Cancer (HNPCC) which is the commonest form. The detection of mutations in the MMR and apc related genes, allows the development of health prevention strategies. Different molecular diagnostic strategies are available for the detection of mutations in these genes, i.e. DGGE, SSCP and direct sequencing. However, deletions and duplications of one or more consecutive exons, which account for around 50% of the total alterations in MMR genes, cannot be detected by PCR based methodologies due to the non quantitative nature of these techniques. The aim of our work has been the standardization of a methodology, called Multiplex Ligation-Dependent Probe Amplification, which allows the detection of genomic deletions and duplications as primary analysis in HNPCC and FAP patients in Argentina. In this case, we inform that the application of MLPA allowed the detection of a missence mutation, without the need for direct sequencing of the complete genes involved. A PCR/RFLP strategy was afterwards designed to detect the C<T change on codon 718 of mlh1 gene in members of the family. For a developing country like Argentina, which has limited resources for genetic diagnosis, this MLPA application has avoided an unaffordable cost as the complete sequencing of all the involved genes. The application of MLPA in our country contributes to improvement in the diagnosis of hereditary CC and allows the development of preventive health interventions.
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Affiliation(s)
- Laura C Gomez
- Laboratory of Cellular and Molecular Biology-IHEM-CONICET, School of Medical Sciences, National University of Cuyo, Mendoza, 5500, Argentina
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Vaughn CP, Lyon E, Samowitz WS. Confirmation of single exon deletions in MLH1 and MSH2 using quantitative polymerase chain reaction. J Mol Diagn 2008; 10:355-60. [PMID: 18556772 DOI: 10.2353/jmoldx.2008.080021] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Deletions of one or more exons in the mismatch repair genes MLH1 and MSH2 have been implicated in a significant fraction of hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Multiplex ligation-dependent probe amplification (MLPA) detection of deletions of multiple sequential exons is widely accepted; however, there is concern over the reliability of MLPA results showing single exon deletions. Given the clinical implications of a diagnosis of Lynch syndrome, it is important to use an alternative technique to confirm single exon deletions. To verify single exon deletions, we developed a SYBR Green-based quantitative polymerase chain reaction (PCR) assay. Clinical DNA samples containing deletions in 33 of the 35 exons in MLH1 and MSH2, previously screened by MLPA, were evaluated by quantitative PCR. Gene dosage ratios were determined by both the relative standard curve method and by the 2(-DeltaDeltaC(T)) method. Deleted exons had gene dosage ratios of 0.4 to 0.6, while nondeleted exons exhibited ratios of 0.8-1.3. We found 100% concordance between the quantitative PCR and MLPA results, including confirmation of all single exon deletions. The 2(-DeltaDeltaC(T)) method was as accurate as using standard curves for the calculation of ratios. Single exon deletions in MLH1 and MSH2 can be verified using quantitative PCR. Assays using this method are simple to design and easy to perform, making them ideal for confirmatory testing.
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Affiliation(s)
- Cecily P Vaughn
- ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA
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40
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Abstract
Hereditary forms of hypertrophic, dilated, restrictive, and right ventricular cardiomyopathies are frequently seen. Patterns of inheritance include autosomal dominant, autosomal recessive, X-linked, and matrilinear. Recognition of the mode of inheritance facilitates proper clinical screening of family members in subsequent generations. Report of successful sequence analysis of the human genome 7 years ago has resulted in widespread translation of genomic information into clinical applications. As technologic advances in high throughput sequence determination continue to evolve, an era of personalized medicine based on genomic data is highly anticipated. Today, clinical genetic testing is available for most monogenic forms of cardiomyopathy and the demand among patients and families is increasing. However, physicians and patients should consider the benefits and limitations of such testing. This review will focus on inherited forms of cardiomyopathy, detailing the currently available genetic tests, as well as benefits, limitations, and possible outcomes of such testing.
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Devlin LA, Graham CA, Price JH, Morrison PJ. Germline MSH6 mutations are more prevalent in endometrial cancer patient cohorts than hereditary non polyposis colorectal cancer cohorts. THE ULSTER MEDICAL JOURNAL 2008; 77:25-30. [PMID: 18269114 PMCID: PMC2397009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
OBJECTIVE To determine and compare the prevalence of MSH6 (a mismatch repair gene) mutations in a cohort of families with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), and in an unselected cohort of endometrial cancer patients (EC). DESIGN Two patient cohorts participated in the study. A cohort of HNPCC families who were known to the Regional Medical Genetics department, and an unselected cohort of patients with a history of EC. All participants received genetic counselling on the implications of molecular testing, and blood was taken for DNA extraction with consent. All samples underwent sequencing and Multiple Ligation probe analysis (MLPA) for mutations in MSH6. POPULATIONS DNA from one hundred and forty-three probands from HNPCC families and 125 patients with EC were included in the study. METHODS Molecular analysis of DNA in all participants from both cohorts for mutations in MSH6. OUTCOME MEASURES Prevalence of pathogenic mutations in MSH6. RESULTS A truncating mutation in MSH6 was identified in 3.8% (95% CI 1.0-9.5%) of patients in the endometrial cancer cohort, and 2.6% (95% CI 0.5-7.4%) of patients in the HNPCC cohort. A missense mutation was identified in 2.9% and 4.4% of the same cohorts respectively. No genomic rearrangements in MSH6 were identified. CONCLUSION MSH6 mutations are more common in EC patients than HNPCC families. Genomic rearrangements do not contribute to a significant proportion of mutations in MSH6, but missense variants are relatively common and their pathogenicity can be uncertain. HNPCC families may be ascertained through an individual presenting with EC, and recognition of these families is important so that appropriate cancer surveillance can be put in place.
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Affiliation(s)
- Lisa A Devlin
- Department of Medical Genetics, Belfast City HospitalBelfast HSC Trust, Belfast, BT9 7AB, United Kingdom
| | - Colin A Graham
- Department of Medical Genetics, Belfast City HospitalBelfast HSC Trust, Belfast, BT9 7AB, United Kingdom
| | - John H Price
- Department of Gynaecological Oncology, Belfast City HospitalBelfast HSC Trust, Belfast, BT9 7AB, United Kingdom
| | - Patrick J Morrison
- Department of Medical Genetics, Belfast City HospitalBelfast HSC Trust, Belfast, BT9 7AB, United Kingdom
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42
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Development of a modular system for detection of genetically modified organisms in food based on ligation-dependent probe amplification. Eur Food Res Technol 2007. [DOI: 10.1007/s00217-007-0790-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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Yanaba K, Nakagawa H, Takeda Y, Koyama N, Sugano K. Muir-Torre syndrome caused by partial duplication of MSH2 gene by Alu-mediated nonhomologous recombination. Br J Dermatol 2007; 158:150-6. [PMID: 17941949 DOI: 10.1111/j.1365-2133.2007.08233.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
We describe a 54-year-old man with a pedicled tumour on the neck. The surgical specimen revealed a sebaceous carcinoma. He belonged to a cancer-prone family susceptible to gastrointestinal cancer. Systemic evaluation for latent malignancies revealed early-stage colonic adenocarcinoma. These findings were compatible with Muir-Torre syndrome (MTS). Microsatellite instability was detected in the sebaceous carcinoma, suggesting a DNA mismatch repair gene mutation. Moreover, duplication of exon 7 generated a nonsense codon at codon 427 of the MSH2 gene causing truncation of MSH2 protein. Immunohistochemical analysis showed diminished MSH2 protein levels in the sebaceous carcinoma and colonic adenocarcinoma. To date, there have been no reports showing duplication of exon 7 of the MSH2 gene in MTS or hereditary nonpolyposis colorectal cancer kindreds. Furthermore, the present case indicates that the dermatologist plays an important role in the diagnosis of MTS and evaluation for latent malignancies.
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Affiliation(s)
- K Yanaba
- Department of Dermatology, The Jikei University School of Medicine, 3-25-8 Nishishimbashi, Minato-ku, Tokyo, Japan.
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Signori E, Massi E, Matera MG, Poscente M, Gravina C, Falcone G, Rosa MA, Rinaldi M, Wuyts W, Seripa D, Dallapiccola B, Fazio VM. A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients. Genes Chromosomes Cancer 2007; 46:470-7. [PMID: 17301954 DOI: 10.1002/gcc.20429] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Multiple osteochondromas (MO), also known as hereditary multiple exostoses (HME), is one of the most common hereditary musculoskeletal diseases in Caucasians (1/50,000) with wide clinical variability and genetic heterogeneity. Two genes have thus far been identified as causing the disease, namely EXT1 and EXT2. Various methods to detect mutations in the EXT genes have been used. Here a cohort of 100 MO patients belonging to unrelated Italian families have been analyzed by single-strand conformation polymorphism (SSCP) analysis or by denaturing high performance liquid chromatography (DHPLC). However, neither of these techniques can detect deletions or duplications of entire exons. Families that were negative at SSCP/DHPLC analysis underwent two-color multiple ligation-dependent probe amplification (MLPA) analysis. By these complementary techniques mutation detection was significantly improved and 26 novel mutations have been revealed as well as 18 previously described mutations to give a total of 44 different mutations. Thus we can conclude that combining MLPA with DHPLC in point-mutations negative MO families, the detection of mutations in EXT genes can significantly improve the identification of both point-mutations and mid-size rearrangements. More important, we were able to characterize all those patients who were negative at the first PCR-based method screening.
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Affiliation(s)
- Emanuela Signori
- Laboratory of Molecular Medicine and Biotechnology, University Campus Bio-Medico School of Medicine and Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy.
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45
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Ewald J, Rodrigue CM, Mourra N, Lefèvre JH, Fléjou JF, Tiret E, Gespach C, Parc YR. Immunohistochemical staining for mismatch repair proteins, and its relevance in the diagnosis of hereditary non-polyposis colorectal cancer. Br J Surg 2007; 94:1020-7. [PMID: 17440950 DOI: 10.1002/bjs.5704] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
BACKGROUND Hereditary non-polyposis colorectal cancer (HNPCC) arises mostly from germline mutations of the mismatch repair genes MSH2 and MLH1. The diagnosis of HNPCC is based on a set of clinical criteria that may be too restrictive to identify all affected patients. Immunohistochemical staining (IHC) for the mismatch repair proteins, MutS homologue 2 (MSH2) and MutL homologue 1 (MLH1), reliably identifies the microsatellite instability phenotype. This study evaluated the ability of IHC to detect germline mutations in an unselected group of patients with colorectal cancer (CRC). METHODS All patients with CRC operated on between July 2000 and March 2003, and demonstrating a loss of protein, were contacted. Following informed consent, searchs for germline mutation and methylation of the promoter were performed on normal and tumoral DNA. RESULTS Thirty patients agreed to participate, four of whom fulfilled the Amsterdam II criteria. Loss of expression of MLH1 was found in 20 patients, and loss of expression of MSH2 in ten patients. Four of the MLH1-deficient patients had a germline MLH1 point mutation (positive predictive value (PPV) 20 (95 per cent confidence interval (c.i.) 2 to 38 per cent) and 11 had promoter methylation. Seven of the MSH2-deficient patients had a germline MSH2 point mutation (PPV 70 (95 per cent c.i. 54 to 96 per cent), and none showed promoter methylation. CONCLUSION MLH1-deficient patients who are young or have a positive family history of cancer should be referred for genetic testing and counselling, whereas MSH2-deficient patients should be counselled in the same way as patients with HNPCC.
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Affiliation(s)
- J Ewald
- Department of Digestive Surgery, Hôpital Saint-Antoine (AP/HP), Université Pierre et Marie Curie, Paris, France
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46
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Aretz S, Stienen D, Uhlhaas S, Stolte M, Entius MM, Loff S, Back W, Kaufmann A, Keller KM, Blaas SH, Siebert R, Vogt S, Spranger S, Holinski-Feder E, Sunde L, Propping P, Friedl W. High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome. J Med Genet 2007; 44:702-9. [PMID: 17873119 PMCID: PMC2752176 DOI: 10.1136/jmg.2007.052506] [Citation(s) in RCA: 150] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
BACKGROUND In patients with juvenile polyposis syndrome (JPS) the frequency of large genomic deletions in the SMAD4 and BMPR1A genes was unknown. METHODS Mutation and phenotype analysis was used in 80 unrelated patients of whom 65 met the clinical criteria for JPS (typical JPS) and 15 were suspected to have JPS. RESULTS By direct sequencing of the two genes, point mutations were identified in 30 patients (46% of typical JPS). Using MLPA, large genomic deletions were found in 14% of all patients with typical JPS (six deletions in SMAD4 and three deletions in BMPR1A). Mutation analysis of the PTEN gene in the remaining 41 mutation negative cases uncovered a point mutation in two patients (5%). SMAD4 mutation carriers had a significantly higher frequency of gastric polyposis (73%) than did patients with BMPR1A mutations (8%) (p<0.001); all seven cases of gastric cancer occurred in families with SMAD4 mutations. SMAD4 mutation carriers with gastric polyps were significantly older at gastroscopy than those without (p<0.001). In 22% of the 23 unrelated SMAD4 mutation carriers, hereditary hemorrhagic telangiectasia (HHT) was also diagnosed clinically. The documented histologic findings encompassed a wide distribution of different polyp types, comparable with that described in hereditary mixed polyposis syndromes (HMPS). CONCLUSIONS Screening for large deletions raised the mutation detection rate to 60% in the 65 patients with typical JPS. A strong genotype-phenotype correlation for gastric polyposis, gastric cancer, and HHT was identified, which should have implications for counselling and surveillance. Histopathological results in hamartomatous polyposis syndromes must be critically interpreted.
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Affiliation(s)
- S Aretz
- Institute of Human Genetics, University of Bonn, Wilhelmstrasse 31, D-53111 Bonn, Germany.
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van Hagen JM, Eussen HJ, van Schooten R, van Der Geest JN, Lagers-van Haselen GC, Wouters CH, De Zeeuw CI, Gille JJ. Comparing Two Diagnostic Laboratory Tests for Williams Syndrome: Fluorescent In Situ Hybridization versus Multiplex Ligation-Dependent Probe Amplification. ACTA ACUST UNITED AC 2007; 11:321-7. [DOI: 10.1089/gte.2007.0007] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Affiliation(s)
- Johanna M. van Hagen
- Department of Clinical Genetics, VU University Medical Center, 1007 MB Amsterdam, The Netherlands
| | | | - Ron van Schooten
- Department of Clinical Genetics, VU University Medical Center, 1007 MB Amsterdam, The Netherlands
| | | | | | - Cokkie H. Wouters
- Department of Clinical Genetics, Erasmus MC, 3000 DR Rotterdam, The Netherlands
| | - Chris I. De Zeeuw
- Department of Neuroscience, Erasmus MC, 3000 CA Rotterdam, The Netherlands
| | - Johan J.P. Gille
- Department of Clinical Genetics, VU University Medical Center, 1007 MB Amsterdam, The Netherlands
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Zidan J, Niessen RC, Laitman Y, Rozeveld D, Hofstra RMW, Friedman E. A novel MSH2 germline mutation in a Druze HNPCC family. Fam Cancer 2007; 7:135-9. [PMID: 17661183 DOI: 10.1007/s10689-007-9157-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2006] [Accepted: 07/15/2007] [Indexed: 01/20/2023]
Abstract
Germline mutations in DNA mismatch repair (DNA-MMR) genes, mainly MLH1, MSH2, and MSH6, underlie Hereditary non-polyposis colorectal cancer (HNPCC) and are mostly family-specific, with few reported founder mutations in MSH2 (Ashkenazim) MLH1 (Finnish). No mutations in colon cancer susceptibility genes have ever been reported in Druze individuals, a Moslem related faith encompassing approximately 1,000,000 individuals worldwide. A novel MSH2 mutation is described in a Druze HNPCC family: a multigenerational family with 10 members in 4 generations affected with colorectal cancer (mean age of diagnosis 46.5 years), two with gastric cancer and one--endometrial cancer. Mutational analysis of the MSH2 gene using denaturing gradient gel electrophoresis (DGGE) and direct sequencing revealed the c.702delA mutation in codon 234 of exon 4 of the MSH2 gene leading to a premature early stop in codon 245, p.Thr234fsX245. Analysis of mutation-carrying or presumed carriers individuals' offspring, revealed 11/42 asymptomatic mutation carriers, age range 17-50 years. The mutation was not present in two additional Druze HNPCC families and 20 Druze sporadic colon cancer patients. This is the first mutation ever reported in a colon cancer susceptibility gene in a Druze family and it appears not to be a founder mutation in Druze individuals with HNPCC.
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Affiliation(s)
- Jamal Zidan
- Oncology Unit, Rivkah Ziv Medical Center, Zefat, Israel
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Karlan BY, Berchuck A, Mutch D. The Role of Genetic Testing for Cancer Susceptibility in Gynecologic Practice. Obstet Gynecol 2007; 110:155-67. [PMID: 17601911 DOI: 10.1097/01.aog.0000269050.79143.84] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Genetic counseling and testing for inherited disorders are part of every obstetrician-gynecologist's practice. Family history, ethnicity, and race are routinely evaluated as a part of the prenatal assessment. The discovery of genes responsible for inherited cancer susceptibility and the wide availability of clinical genetic testing for mutations in these genes have made similar assessments an integral part of gynecologic practice as well. The indications for genetic testing for mutations in BRCA1, BRCA2, and the mismatch repair genes responsible for the hereditary nonpolyposis colon cancer (HNPCC) syndrome need to be individualized. As in obstetrics, genetic counseling can provide critical assessment of the family history to help determine the likelihood of an inherited cancer susceptibility syndrome and the appropriateness of genetic testing. The subsequent clinical recommendations for mutation carriers need to take into account the patient's age, desire for future childbearing, and other medical history when prescribing screening interventions or prophylactic surgery. Practical applications of genetic testing for cancer susceptibility have the ability to reduce the burden of hereditary cancers by saving lives, decreasing medical morbidities, and reducing psychological stress.
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Affiliation(s)
- Beth Y Karlan
- Women's Cancer Research Institute, Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of Los Angeles, Los Angeles, California 90048, USA.
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50
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Velasco E, Infante M, Durán M, Pérez-Cabornero L, Sanz DJ, Esteban-Cardeñosa E, Miner C. Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes. Nat Protoc 2007; 2:237-46. [PMID: 17401359 DOI: 10.1038/nprot.2006.482] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7-8 h.
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Affiliation(s)
- Eladio Velasco
- Laboratorio de Genética del Cáncer, Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid, Valladolid, Spain.
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