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Cao WM, Gao Y, Yang HJ, Xie SN, Ding XW, Pan ZW, Ye WW, Wang XJ. Novel germline mutations and unclassified variants of BRCA1 and BRCA2 genes in Chinese women with familial breast/ovarian cancer. BMC Cancer 2016; 16:64. [PMID: 26852015 PMCID: PMC4744435 DOI: 10.1186/s12885-016-2107-6] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2014] [Accepted: 02/01/2016] [Indexed: 01/21/2023] Open
Abstract
BACKGROUND Germline mutations in the BRCA1 and BRCA2 genes greatly increase a woman's risk of developing breast and/or ovarian cancer. The prevalence and distribution of such mutations differ across races/ethnicities. Several studies have investigated Chinese women with high-risk breast cancer, but the full spectrum of the mutations in these two genes remains unclear. METHODS In this study, 133 unrelated Chinese women with familial breast/ovarian cancer living in Zhejiang, eastern China, were enrolled between the years 2008 and 2014. The complete coding regions and exon-intron boundaries of BRCA1 and BRCA2 were screened by PCR-sequencing assay. Haplotype analysis was performed to confirm BRCA1 and BRCA2 founder mutations. In silico predictions were performed to identify the non-synonymous amino acid changes that were likely to disrupt the functions of BRCA1 and BRCA2. RESULTS A total of 23 deleterious mutations were detected in the two genes in 31 familial breast/ovarian cancer patients with a total mutation frequency of 23.3% (31/133). The highest frequency of 50.0% (8/16) was found in breast cancer patients with a history of ovarian cancer. The frequencies of BRCA1 and BRCA2 mutations were 13.5 % (18/133) and 9.8% (13/133), respectively. We identified five novel deleterious mutations (c.3295delC, c.3780_3781delAG, c.4063_4066delAATC, c.5161 > T and c.5173insA) in BRCA1 and seven (c.1-40delGA, c.4487delC, c.469_473delAAGTC, c.5495delC, c.6141T > A, c.6359C > G and c.7588C > T) in BRCA2, which accounted for 52.2% (12/23) of the total mutations. Six recurrent mutations were found, including four (c.3780_3781delAG, c.5154G > A, c.5468-1del8 and c.5470_5477del8) in BRCA1 and two (c.3109C > T and c.5682C > G) in BRCA2. Two recurrent BRCA1 mutations (c.5154G > A and c.5468-1del8) were identified as putative founder mutations. We also found 11 unclassified variants, and nine of these are novel. The possibility was that each of the non-synonymous amino acid changes would disrupt the function of BRCA1 and BRCA2 varied according to the different algorithms used. CONCLUSIONS BRCA1 and BRCA2 mutations accounted for a considerable proportion of hereditary breast/ovarian cancer patients from eastern China and the spectrum of the mutations of these two genes exhibited some unique features. The two BRCA1 putative founder mutations may provide a cost-effective option to screen Chinese population, while founder effects of the two mutations should be investigated in a lager sample size of patients.
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Affiliation(s)
- Wen-Ming Cao
- />Department of Medical Oncology, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou, 310022 China
| | - Yun Gao
- />Institute of Cancer Research, Zhejiang Cancer Hospital, Hangzhou, 310022 China
| | - Hong-Jian Yang
- />Department of Breast Cancer Surgery, Zhejiang Cancer Hospital, Hangzhou, 310022 China
| | - Shang-Nao Xie
- />Department of Breast Cancer Surgery, Zhejiang Cancer Hospital, Hangzhou, 310022 China
| | - Xiao-Wen Ding
- />Department of Breast Cancer Surgery, Zhejiang Cancer Hospital, Hangzhou, 310022 China
| | - Zhi-Wen Pan
- />Department of Clinical Laboratory, Zhejiang Cancer Hospital, Hangzhou, 310022 China
| | - Wei-Wu Ye
- />Department of Medical Oncology, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou, 310022 China
| | - Xiao-Jia Wang
- />Department of Medical Oncology, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou, 310022 China
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Bausch B, Wellner U, Peyre M, Boedeker CC, Hes FJ, Anglani M, de Campos JM, Kanno H, Maher ER, Krauss T, Sansó G, Barontini M, Letizia C, Hader C, Schiavi F, Zanoletti E, Suárez C, Offergeld C, Malinoc A, Zschiedrich S, Glasker S, Bobin S, Sterkers O, Ba Huy PT, Giraud S, Links T, Eng C, Opocher G, Richard S, Neumann HPH. Characterization of endolymphatic sac tumors and von Hippel-Lindau disease in the International Endolymphatic Sac Tumor Registry. Head Neck 2015; 38 Suppl 1:E673-9. [DOI: 10.1002/hed.24067] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2014] [Revised: 01/01/2015] [Accepted: 04/10/2015] [Indexed: 11/09/2022] Open
Affiliation(s)
- Birke Bausch
- Second Department of Medicine; Albert-Ludwigs-University of Freiburg; Freiburg Germany
| | - Ulrich Wellner
- Department of Surgery; University Hospital Schleswig-Holstein, Campus Luebeck; Luebeck Germany
| | - Mathieu Peyre
- Center Expert National Cancers Rares PREDIR, AP-HP INCa, Hôpital de Bicêtre; Le Kremlin-Bicêtre France
- Génétique Oncologique EPHE, INSERM U 753, Faculté de Médecine Paris Sud and Institut de Cancérologie Gustave Roussy, Villejuif, France and Service de Neurochirurgie, AP-HP; Hôpital Beaujon Clichy France
| | - Carsten C. Boedeker
- Department of Otorhinolaryngology; University Medical Center, Albert-Ludwigs-University; Freiburg
- HELIOS Hanseklinikum Stralsund; Stralsund Germany
| | - Frederik J. Hes
- Department of Clinical Genetics; Leiden University Medical Center; Leiden The Netherlands
| | | | - Jose M. de Campos
- Department of Neurosurgery; IIS - Fundación Jiménez Díaz. UAM; Madrid Spain
| | - Hiroshi Kanno
- Department of Neurosurgery; Yokohama City University; Yokohama Japan
| | - Eamonn R. Maher
- Department of Medical Genetics; University of Cambridge and NIHR Cambridge Biomedical Research Center; Cambridge United Kingdom
| | - Tobias Krauss
- Department of Radiology; Albert-Ludwigs-University of Freiburg; Freiburg Germany
| | - Gabriela Sansó
- Centro de Investigaciones Endocrinológicas (CONICET), Hospital de Niños “R. Gutiérrez,”; Buenos Aires Argentina
| | - Marta Barontini
- Centro de Investigaciones Endocrinológicas (CONICET), Hospital de Niños “R. Gutiérrez,”; Buenos Aires Argentina
| | - Claudio Letizia
- Department of Internal Medicine and Medical Specialities; University of Rome “Sapienza,”; Rome Italy
| | - Claudia Hader
- Department of Neuroradiology; Albert-Ludwigs-University; Freiburg Germany
- Department of Radiology and Nuclear Medicine; Kantonsspital St. Gallen Switzerland
| | - Francesca Schiavi
- Familial Cancer Clinic and Oncoendocrinology; Veneto Institute of Oncology IRCCS; Padova Italy
| | - Elisabetta Zanoletti
- Otolaryngology; Department of Otosurgery - Neurosciences; University Hospital of Padova; Padova Italy
| | - Carlos Suárez
- Department of Otolaryngology; Hospital Universitario Central de Asturias and IUOPA, Universidad de Oviedo; Spain
| | - Christian Offergeld
- Department of Otorhinolaryngology; University Medical Center, Albert-Ludwigs-University; Freiburg
| | - Angelica Malinoc
- Department of Nephrology and Hypertension; Albert-Ludwigs-University; Freiburg Germany
| | - Stefan Zschiedrich
- Department of Nephrology and Hypertension; Albert-Ludwigs-University; Freiburg Germany
| | - Sven Glasker
- Department of Neurosurgery; Albert-Ludwigs-University; Freiburg Germany
| | - Serge Bobin
- Service d'ORL, AP-HP, Hôpital de Bicêtre; Le Kremlin-Bicêtre France
| | - Olivier Sterkers
- AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Unité Otologie, Implants auditifs et Chirurgie de la base du crâne; Paris France
- Université Paris, Pierre et Marie Curie; France
| | | | - Sophie Giraud
- Center Expert National Cancers Rares PREDIR, AP-HP INCa, Hôpital de Bicêtre; Le Kremlin-Bicêtre France
- Laboratoire de Génétique, Hôpital Edouard Herriot; Lyon France
| | - Thera Links
- Department of Endocrinology; Groningen University Medical Center; Groningen The Netherlands
| | - Charis Eng
- Genomic Medicine Institute, Lerner Research Institute and Taussig Cancer Institute, Cleveland Clinic; Cleveland Ohio
| | - Giuseppe Opocher
- Familial Cancer Clinic and Oncoendocrinology; Veneto Institute of Oncology IRCCS; Padova Italy
| | - Stephane Richard
- Center Expert National Cancers Rares PREDIR, AP-HP INCa, Hôpital de Bicêtre; Le Kremlin-Bicêtre France
- Génétique Oncologique EPHE, INSERM U 753, Faculté de Médecine Paris Sud and Institut de Cancérologie Gustave Roussy, Villejuif, France and Service de Neurochirurgie, AP-HP; Hôpital Beaujon Clichy France
| | - Hartmut P. H. Neumann
- Department of Nephrology and Hypertension; Albert-Ludwigs-University; Freiburg Germany
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Gossage L, Murtaza M, Slatter AF, Lichtenstein CP, Warren A, Haynes B, Marass F, Roberts I, Shanahan SJ, Claas A, Dunham A, May AP, Rosenfeld N, Forshew T, Eisen T. Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma. Genes Chromosomes Cancer 2014; 53:38-51. [PMID: 24166983 DOI: 10.1002/gcc.22116] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2013] [Accepted: 09/16/2013] [Indexed: 12/21/2022] Open
Abstract
VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.
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Affiliation(s)
- Lucy Gossage
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK
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Abstract
Breast cancer is the most common malignancy among women and has a strong genetic background. So far, 13 breast cancer susceptibility genes of high or moderate penetrance have been identified. This review summarizes findings on these genes in Han Chinese. BRCA1 and BRCA2 are the 2 most important susceptibility genes. They have a relatively low mutation rate, and the most frequent sites of mutation are in exon 11. Frameshift mutations are the main type of mutation. Founder mutations may also exist, and BRCA-associated breast cancer has specific clinicopathologic characteristics. TP53 and PALB2 are relatively rare susceptibility genes. The relationship between the other 9 genes and breast cancer has not been fully elucidated. At present, the mutation spectrum for these susceptibility genes is not well understood in the Chinese population, and there are few reports on prognosis and clinical intervention in high-risk populations. Therefore, the true value of genetic counseling for breast cancer has yet to be realized. This article reviews studies of hereditary breast cancer in the Han Chinese population, highlights potential inadequacies, and provides a foundation for genetic counseling for breast cancer in China.
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Affiliation(s)
- Wenming Cao
- Institute of Cell Biology, Zhejiang University, Hangzhou, China
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Cao W, Wang X, Gao Y, Yang H, Li JC. BRCA1 germ-line mutations and tumor characteristics in eastern Chinese women with familial breast cancer. Anat Rec (Hoboken) 2012; 296:273-8. [PMID: 23175448 DOI: 10.1002/ar.22628] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2012] [Accepted: 10/30/2012] [Indexed: 01/16/2023]
Abstract
Although several studies detected the BRCA1 germ-line mutations in Chinese women with familial breast cancer, most of them did not employ conventional full gene sequencing, especially in eastern China. In addition, the clinicopathological features of BRCA1-associated breast cancer in Chinese women were not well investigated. In this study, we screened the complete coding regions and exon-intron boundaries of BRCA1 by polymerase chain reaction (PCR)-sequencing assay. Immunohistochemistry analyses were performed on tumor samples to detect the expression of estrogen receptor (ER), progesterone receptor (PR), P53, and human epidermal growth factor receptor-2 (HER-2). Breast cancer patients having one or more affected relatives referred from the Zhejiang Cancer Hospital, eastern China during 2008-2011 were selected for the study. A total of 62 familial breast cancer patients received the BRCA1 germ-line mutation screening. Five deleterious mutations were detected in this cohort. The mutation rate was 11.3% (7/62). We found two novel mutations (3414delC and 5,280 C > T) and two recurrent mutations (5,273 G > A and 5589del8). BRCA1 mutation tumors tended to be negative for ER, PR, and HER-2, and exhibited high histological grade compared with tumors without BRCA1 mutations. Our study suggests that recurrent mutations may exist in eastern Chinese women with familial breast cancer and PCR-sequencing assay is a useful tool to screen these mutations. It also suggests that BRCA1-associated breast cancers in Chinese women exhibit an aggressive phenotype.
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Affiliation(s)
- Wenming Cao
- Institute of Cell Biology, Zhejiang University, Hangzhou 310058, China
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Wu P, Zhang N, Wang X, Ning X, Li T, Bu D, Gong K. Family history of von Hippel-Lindau disease was uncommon in Chinese patients: suggesting the higher frequency of de novo mutations in VHL gene in these patients. J Hum Genet 2012; 57:238-43. [PMID: 22357542 DOI: 10.1038/jhg.2012.10] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome caused by germline mutations in VHL tumor suppressor gene. It is characterized by hemangioblastoma in central nervous system and retina, renal cell carcinoma or cyst, pheochromocytoma, pancreatic cyst and tumor, endolymphatic-sac tumor, and papillary cystadenoma in epididymis and broad ligament. Here, we used PCR-direct sequencing and universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR) to detect VHL mutations in 16 patients clinically diagnosed with VHL disease. PCR-direct sequencing detected 12 germline mutations (75%, 12/16), in which a novel mutation of c.451A>T/p.Ile151Phe found in one proband had not been reported previously. UPQFM-PCR found two large deletions (12.5%, 2/16). The two remaining patients carried non-typical disease-causing mutations, including one silent mutation (c.481C>A/p.Arg161Arg) and one mutation in 3'-UTR (c.642+70C>A). Remarkably, 56.3% (9/16) probands did not have family history of VHL disease, suggesting the higher frequency of de novo mutations in Chinese patients. We also summarized Chinese VHL disease patients with VHL mutation findings published in the literature to provide information about the spectrum of VHL mutations in Chinese VHL disease patients.
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Affiliation(s)
- Pengjie Wu
- Institute of Urology, Peking University, Beijing, PR China
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Chacon‐Camacho OF, Rodriguez‐Dennen F, Camacho‐Molina A, Rasmussen A, Alonso‐Vilatela E, Zenteno JC. Clinical and molecular features of familial and sporadic cases of von Hippel‐Lindau disease from Mexico. Clin Exp Ophthalmol 2010; 38:277-83. [DOI: 10.1111/j.1442-9071.2010.02241.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
| | | | | | - Astrid Rasmussen
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA,
| | - Elisa Alonso‐Vilatela
- Department of Neurogenetics and Molecular Biology, Instituto Nacional de Neurología y Neurocirugía ‘Manuel Velasco Suarez’, Mexico City, Mexico, and
| | - Juan C Zenteno
- Department of Genetics,
- Department of Biochemistry, Faculty of Medicine, National University Autonomous of Mexico, Mexico City, Mexico
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8
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Hydrolysis of DNA and its molecular components in the dry state. Forensic Sci Int Genet 2010; 4:168-77. [DOI: 10.1016/j.fsigen.2009.08.007] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2009] [Revised: 08/09/2009] [Accepted: 08/11/2009] [Indexed: 11/19/2022]
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Kim WT, Ham WS, Ju HJ, Lee JS, Lee JS, Choi YD. Clinical characteristics of renal cell carcinoma in Korean patients with von Hippel-Lindau disease compared to sporadic bilateral or multifocal renal cell carcinoma. J Korean Med Sci 2009; 24:1145-9. [PMID: 19949673 PMCID: PMC2775865 DOI: 10.3346/jkms.2009.24.6.1145] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2008] [Accepted: 01/20/2009] [Indexed: 12/02/2022] Open
Abstract
This study was done to analyze the clinical characteristics of renal cell carcinoma (RCC) in Korean patients with von Hippel-Lindau (VHL) disease. Between January 1996 and July 2008, 1,514 patients were diagnosed with RCC and 24 patients were diagnosed with VHL disease at our institute. We analyzed the clinical characteristics of the 24 patients diagnosed with VHL. The mean age of patients with VHL was 39.2+/-12.6 yr; the mean age of patients with both VHL and RCC was 42.5+/-10.3 yr. Among the 24 patients with VHL, 7 patients had retinal angiomas, 11 had RCC, 16 had renal lesions, 18 had pancreatic lesions and 21 had cerebellar hemangioblastomas. There was no significant difference between survival rates of patients with VHL alone and those with VHL and RCC. However, cancer-specific survival rates were significantly different between patients with both VHL and RCC and patients with sporadic bilateral or multifocal RCC. In our Korean study, the incidence of RCC in patients with VHL disease is 45.8% and the incidence of VHL disease in patients with RCC is 0.73%. Due to the low overall incidence of VHL in Korea, extended multi-institutional studies are needed to establish the true characteristics of VHL disease.
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Affiliation(s)
- Won Tae Kim
- Department of Urology & Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
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Denaturing High Performance Liquid Chromatography Detection of SDHB, SDHD, and VHL Germline Mutations in Pheochromocytoma. J Surg Res 2009; 157:55-62. [DOI: 10.1016/j.jss.2008.07.043] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2008] [Revised: 07/21/2008] [Accepted: 07/31/2008] [Indexed: 11/24/2022]
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Rohlin A, Wernersson J, Engwall Y, Wiklund L, Björk J, Nordling M. Parallel sequencing used in detection of mosaic mutations: comparison with four diagnostic DNA screening techniques. Hum Mutat 2009; 30:1012-20. [PMID: 19347965 DOI: 10.1002/humu.20980] [Citation(s) in RCA: 138] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments.
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Affiliation(s)
- Anna Rohlin
- Department of Clinical Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden
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Cao AY, Huang J, Hu Z, Li WF, Ma ZL, Tang LL, Zhang B, Su FX, Zhou J, Di GH, Shen KW, Wu J, Lu JS, Luo JM, Yuan WT, Shen ZZ, Huang W, Shao ZM. Mutation analysis of BRIP1/BACH1 in BRCA1/BRCA2 negative Chinese women with early onset breast cancer or affected relatives. Breast Cancer Res Treat 2008; 115:51-5. [PMID: 18483852 DOI: 10.1007/s10549-008-0052-z] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2008] [Accepted: 05/01/2008] [Indexed: 10/22/2022]
Abstract
The proper interaction between BRIP1/BACH1 and BRCA1 protein has been found to be crucial for BRCA1-mediated DNA double-strand break repair and BRIP1/BACH1 mutations were estimated to confer a relative risk for breast cancer of 2.0 in western populations. In Chinese population, BRCA1 mutations could explain a relatively large proportion of inherited breast cancer cases in comparison with BRCA2 mutations, which probably deduced a hypothesis that those genes involved in BRCA1-mediated DNA repair pathway might play a more significant role in the etiology of Chinese breast cancer. To investigate the contribution of BRIP1/BACH1 mutations to the predisposition of Chinese non-BRCA1/BRCA2 hereditary breast cancer, we screened all the coding exons and adjacent intronic splice junction regions of BRIP1/BACH1 in 357 Chinese women with early-onset breast cancer or affected relatives from five different breast disease clinical centers in China, using PCR-DHPLC and DNA sequencing analysis. Some genetic variants identified in the cases were then studied in 864 normal controls with no personal or family history of breast cancer. We found no protein-truncated mutations in our population, while a novel recurrent non-synonymous variant, Q944E, was detected in two independent families in contrast with none in the controls, interestingly, this alteration occurs in the BRCA1 binding domain of the BACH1 protein. Then a further study performed on the two mutation positive families revealed the partial co-segregation of this mutation allele with cancer. The novel alteration Q944E identified in our study possibly represents a rare disease-related allele, nevertheless functional analysis is still warranted to resolve the ability of this altered BACH1 protein to bind BRCA1. Altogether, the results of our study indicated that germline mutations in BRIP1/BACH were extremely rare in Chinese population and there was no evidence for the recommendation of BRIP1/BACH1 for genetic testing in Chinese.
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Affiliation(s)
- A-Yong Cao
- Department of Oncology, Breast Cancer Institute, Cancer Hospital/Cancer Institute, Shanghai Medical College, Institutes of Biomedical Science, Fudan University, Shanghai, People's Republic of China
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The prevalence of PALB2 germline mutations in BRCA1/BRCA2 negative Chinese women with early onset breast cancer or affected relatives. Breast Cancer Res Treat 2008; 114:457-62. [PMID: 18446436 DOI: 10.1007/s10549-008-0036-z] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/04/2008] [Indexed: 12/30/2022]
Abstract
PALB2 has been recently identified as breast cancer susceptibility gene in western populations. To investigate the contribution of PALB2 mutations to Chinese non-BRCA1/BRCA2 hereditary breast cancer, we screened all coding exons and intron-exon boundaries of PALB2 in 360 Chinese women with early-onset breast cancer or affected relatives from five breast disease clinical centers in China by utilizing PCR-DHPLC and DNA sequencing analysis. Some genetic variants identified in the cases were then studied in 864 normal controls with no personal or family history of breast cancer. Two protein-truncating PALB2 mutations, 751C>T and 1050_1051delAAinsTCT, were identified in three separate families, and 751C>T was a recurrent mutation. Neither of them, however, were present in the controls (P=0.025). All the truncating mutations occurred in exon 4 of PALB2, and there were still three unclassified variants were detected in the same fragment. We found that exon 4 accounted for 44.1% (15/34) of the person-times carrying with any variant in our study. PALB2 mutations were responsible for approximately 1% of Chinese women with early-onset breast cancer and affected relatives. Our results suggested that a detection of exon 4 before the assay of the whole PALB2 gene might be a cost-effective approach to the screening of Chinese population.
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Kim WT, Ham WS, Park SJ, Kim SW, Lee JS, Lee JS, Ju HJ, Choi YD. Clinical Characteristics of Renal Cell Carcinoma in Korean Patients with von Hippel-Lindau Disease. Korean J Urol 2008. [DOI: 10.4111/kju.2008.49.10.863] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Affiliation(s)
- Won Tae Kim
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Won Sik Ham
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Sung Jin Park
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Sang Woon Kim
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Jin sung Lee
- Department of Clinical Genetics, Yonsei University College of Medicine, Seoul, Korea
| | - Jin Sun Lee
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Hee Jeong Ju
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Young Deuk Choi
- Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
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Li WF, Hu Z, Rao NY, Song CG, Zhang B, Cao MZ, Su FX, Wang YS, He PQ, Di GH, Shen KW, Wu J, Lu JS, Luo JM, Liu XY, Zhou J, Wang L, Zhao L, Liu YB, Yuan WT, Yang L, Shen ZZ, Huang W, Shao ZM. The prevalence of BRCA1 and BRCA2 germline mutations in high-risk breast cancer patients of Chinese Han nationality: two recurrent mutations were identified. Breast Cancer Res Treat 2007; 110:99-109. [PMID: 17851763 DOI: 10.1007/s10549-007-9708-3] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2007] [Accepted: 07/18/2007] [Indexed: 02/06/2023]
Abstract
To have an overview of the role of BRCA1 and BRCA2 genes among Chinese high-risk breast cancer patients, we analyzed 489 such high-risk breast cancer patients from four breast disease clinical centers in China, by using PCR-DHPLC or SSCP-DNA sequencing analysis. Allelotype analysis was done at five short tandem repeat (STR) markers in or adjacent to BRCA1 on the recurrent mutation carriers. For those analyzed both genes, 8.7% of early-onset breast cancer cases and 12.9% of familial breast cancer cases had a BRCA1 or BRCA2 mutation, as compared with the 26.1% of cases with both early-onset breast cancer and affected relatives. For those reporting malignancy family history other than breast/ovarian cancer, the prevalence of BRCA1/2 mutation is about 20.5%, and it was significantly higher than the patients only with family history of breast/ovarian cancer (P = 0.02). The family history of ovarian cancer (26.7% vs. 11.9%) and stomach cancer (23.8% vs. 11.8%) doubled the incidence of BRCA1/2, but the difference did not reach the statistical significance. Two recurrent mutations in BRCA1, 1100delAT and 5589del8, were identified. The recurrent mutations account for 34.8% BRCA1 mutations in our series. Similar allelotypes were detected in most STR status for those harboring the same mutations. The BRCA1 associated tumors were more likely to exhibit a high tumor grade, negative C-erbB-2/neu status and triple negative (ER, PgR and C-erbB-2/neu negative) status (P < 0.05). We recommended the BRCA1 and BRCA2 genetic analysis could be done for high-risk breast cancer patient in Chinese population, especially for those with both early-onset breast cancer and affected relatives. There may be some degree of shared ancestry for the two recurrent BRCA1 mutations in Chinese.
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Affiliation(s)
- Wen-Feng Li
- Department of Oncology, Breast Cancer Institute, Cancer Hospital/Cancer Institute, Institutes of Biomedical Science, Shanghai Medical College, Fudan University, 270 Dong'an Road, Shanghai 200032, PR China
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16
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Pfendner EG, Vanakker OM, Terry SF, Vourthis S, McAndrew PE, McClain MR, Fratta S, Marais AS, Hariri S, Coucke PJ, Ramsay M, Viljoen D, Terry PF, De Paepe A, Uitto J, Bercovitch LG. Mutation detection in the ABCC6 gene and genotype-phenotype analysis in a large international case series affected by pseudoxanthoma elasticum. J Med Genet 2007; 44:621-8. [PMID: 17617515 PMCID: PMC2597973 DOI: 10.1136/jmg.2007.051094] [Citation(s) in RCA: 151] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
BACKGROUND Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder with considerable phenotypic variability, mainly affects the eyes, skin and cardiovascular system, characterised by dystrophic mineralization of connective tissues. It is caused by mutations in the ABCC6 (ATP binding cassette family C member 6) gene, which encodes MRP6 (multidrug resistance-associated protein 6). OBJECTIVE To investigate the mutation spectrum of ABCC6 and possible genotype-phenotype correlations. METHODS Mutation data were collected on an international case series of 270 patients with PXE (239 probands, 31 affected family members). A denaturing high-performance liquid chromatography-based assay was developed to screen for mutations in all 31 exons, eliminating pseudogene coamplification. In 134 patients with a known phenotype and both mutations identified, genotype-phenotype correlations were assessed. RESULTS In total, 316 mutant alleles in ABCC6, including 39 novel mutations, were identified in 239 probands. Mutations were found to cluster in exons 24 and 28, corresponding to the second nucleotide-binding fold and the last intracellular domain of the protein. Together with the recurrent R1141X and del23-29 mutations, these mutations accounted for 71.5% of the total individual mutations identified. Genotype-phenotype analysis failed to reveal a significant correlation between the types of mutations identified or their predicted effect on the expression of the protein and the age of onset and severity of the disease. CONCLUSIONS This study emphasises the principal role of ABCC6 mutations in the pathogenesis of PXE, but the reasons for phenotypic variability remain to be explored.
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Affiliation(s)
- Ellen G Pfendner
- GeneDx Inc., 207 Perry Parkway, Gaithersburg, Maryland 20877, USA.
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Xu L, Evans J, Ling T, Nye K, Hawkey P. Rapid genotyping of CTX-M extended-spectrum beta-lactamases by denaturing high-performance liquid chromatography. Antimicrob Agents Chemother 2007; 51:1446-54. [PMID: 17210774 PMCID: PMC1855489 DOI: 10.1128/aac.01088-06] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial beta-lactamase genes. The technique was specifically developed to genotype members of all blaCTX-M DNA homology groups. Thirteen well-defined blaCTX-M extended-spectrum beta-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of blaCTX-M genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 blaCTX-M ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of blaCTX-M-producing ESBLs and has great potential for determining the clinical relevance of different and new blaCTX-M genotypes, as well as for epidemiological studies and surveillance programs.
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Affiliation(s)
- Li Xu
- Health Protection Agency, West Midlands Public Health Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham B9 5SS, United Kingdom
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Corbo RM, Prévost M, Raussens V, Gambina G, Moretto G, Scacchi R. Structural and phylogenetic approaches to assess the significance of human Apolipoprotein E variation. Mol Genet Metab 2006; 89:261-9. [PMID: 16621646 DOI: 10.1016/j.ymgme.2006.02.015] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2006] [Accepted: 02/27/2006] [Indexed: 11/26/2022]
Abstract
Apolipoprotein E (APOE) is an important gene whose common polymorphism, and precisely the e *4 allele, has been reportedly associated with some disorders, including Alzheimer's disease (AD) and coronary artery disease. In the course of previous surveys on AD patients and healthy individuals some rare variants were detected by means of Isoelectric focusing and denaturing high-performance liquid chromatography techniques. After a mutation in a gene is identified, the problem arises to understand its effective significance. Structure modelling and phylogenetic analysis methods are widely used to establish the possible deleterious effect of mutations. In this study their usefulness in the analysis of APOE variants was evaluated. The two combined methods provided helpful indications for distinguishing between mutations possibly involved in AD susceptibility and not deleterious mutations.
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Affiliation(s)
- Rosa Maria Corbo
- Department of Genetics and Molecular Biology, University La Sapienza, Rome, Italy
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Chen JM, Férec C, Cooper DN. A systematic analysis of disease-associated variants in the 3' regulatory regions of human protein-coding genes II: the importance of mRNA secondary structure in assessing the functionality of 3' UTR variants. Hum Genet 2006; 120:301-33. [PMID: 16807757 DOI: 10.1007/s00439-006-0218-x] [Citation(s) in RCA: 103] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2006] [Accepted: 05/29/2006] [Indexed: 12/13/2022]
Abstract
In an attempt both to catalogue 3' regulatory region (3' RR)-mediated disease and to improve our understanding of the structure and function of the 3' RR, we have performed a systematic analysis of disease-associated variants in the 3' RRs of human protein-coding genes. We have previously analysed the variants that have occurred in two specific domains/motifs of the 3' untranslated region (3' UTR) as well as in the 3' flanking region. Here we have focused upon 83 known variants within the upstream sequence (USS; between the translational termination codon and the upstream core polyadenylation signal sequence) of the 3' UTR. To place these variants in their proper context, we first performed a comprehensive survey of known cis-regulatory elements within the USS and the mechanisms by which they effect post-transcriptional gene regulation. Although this survey supports the view that RNA regulatory elements function within the context of specific secondary structures, there are no general rules governing how secondary structure might exert its influence. We have therefore addressed this question by systematically evaluating both functional and non-functional (based upon in vitro reporter gene and/or electrophoretic mobility shift assay data) USS variant-containing sequences against known cis-regulatory motifs within the context of predicted RNA secondary structures. This has allowed us not only to establish a reliable and objective means to perform secondary structure prediction but also to identify consistent patterns of secondary structural change that could potentiate the discrimination of functional USS variants from their non-functional counterparts. The resulting rules were then used to infer potential functionality in the case of some of the remaining functionally uncharacterized USS variants, from their predicted secondary structures. This not only led us to identify further patterns of secondary structural change but also several potential novel cis-regulatory motifs within the 3' UTRs studied.
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20
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Culiat CT, Klebig ML, Liu Z, Monroe H, Stanford B, Desai J, Tandan S, Hughes L, Kerley MK, Carpenter DA, Johnson DK, Rinchik EM, Li Q. Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7. Mamm Genome 2005; 16:555-66. [PMID: 16180137 DOI: 10.1007/s00335-005-0032-0] [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: 10/25/2022]
Abstract
We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.
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Affiliation(s)
- Cymbeline T Culiat
- Life Sciences Division, Oak Ridge National Laboratory, Bethel Valley Road, P.O. Box 2008, Oak Ridge, Tennessee, 37831-6445, USA.
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Benusiglio PR, Lesueur F, Luccarini C, McIntosh J, Luben RN, Smith P, Dunning A, Easton DF, Ponder BAJ, Pharoah PD. Common variation in EMSY and risk of breast and ovarian cancer: a case-control study using HapMap tagging SNPs. BMC Cancer 2005; 5:81. [PMID: 16029503 PMCID: PMC1185523 DOI: 10.1186/1471-2407-5-81] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2005] [Accepted: 07/19/2005] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND EMSY could be involved in low-level susceptibility to breast and ovarian cancer. Gene amplification is seen in a proportion of breast and ovarian tumours and correlates with poor prognosis in breast cancer patients. Furthermore, the EMSY protein silences a transcription activation domain in BRCA2 exon 3. METHODS We used a genetic association study design to determine if common genetic variation (frequency > or = 5%) in EMSY was associated with breast or ovarian cancer risk in the British population. Haplotype tagging single-nucleotide polymorphisms (htSNPs) were selected from the HapMap database and genotyped using Taqman in two large study sets of white British women (n [breast set] = 2343 cases and 2284 controls, n [ovarian set] = 864 cases and 864 controls). HapMap data might be insufficient to tag genetic variation in EMSY comprehensively. We therefore screened the gene promoter and coding sequences with denaturing high performance liquid chromatography in order to identify additional SNPs that are most likely to be functional. RESULTS HapMap data on 22 SNPs show that 4 htSNPs tag 4 common haplotypes: rs2282611 (5'up t > g), rs4245443 (IVS7 g > a), rs2513511 (IVS16 a > g), rs2155220 (3'down c > t). We observed no association between any of the genotypes or associated haplotypes and breast or ovarian cancer risk. Seventeen out of the 18 remaining HapMap polymorphisms (94%) were well tagged by the 4 selected htSNPs (r2s > 0.8). Genotype frequencies for two further SNPs identified by screening and located near exon-intron boundaries, rs2508740 (IVS9 a > g) and rs11600501 (IVS10 c > t), were also similar in cases and controls. In order to simulate unidentified SNPs, we performed the leave-one-out cross-validation procedure on the HapMap data; over 95% of the common genetic variation was well represented by tagging polymorphisms. We are therefore likely to have tagged any common, functional variants present in our population. CONCLUSION We found no association between common genetic variation in EMSY and risk of breast or ovarian cancer in two large study sets of white British women.
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Affiliation(s)
- Patrick R Benusiglio
- Strangeways Research Laboratory, Department of Oncology, University of Cambridge, Worts Causeway, Cambridge CB1 8RN, UK.
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Yu B, Sawyer NA, Caramins M, Yuan ZG, Saunderson RB, Pamphlett R, Richmond DR, Jeremy RW, Trent RJ. Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease. J Clin Pathol 2005; 58:479-85. [PMID: 15858117 PMCID: PMC1770671 DOI: 10.1136/jcp.2004.021642] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
AIMS To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.
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Affiliation(s)
- B Yu
- Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital and Central Clinical School, The University of Sydney, Missenden Road, Camperdown, NSW 2050, Australia.
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23
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Lu C, Xu HM, Ren Q, Ao Y, Wang ZN, Ao X, Jiang L, Luo Y, Zhang X. Somatic mutation analysis of p53 and ST7 tumor suppressor genes in gastric carcinoma by DHPLC. World J Gastroenterol 2003; 9:2662-5. [PMID: 14669308 PMCID: PMC4612027 DOI: 10.3748/wjg.v9.i12.2662] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To verify the effectiveness of denaturing high-performance liquid chromatography (DHPLC) in detecting somatic mutation of p53 gene in gastric carcinoma tissues. The superiority of this method has been proved in the detection of germline mutations, but it was not very affirmative with respect to somatic mutations in tumor specimens. ST7 gene, a candidate tumor suppressor gene identified recently at human chromosome 7q31.1, was also detected because LOH at this site has also been widely reported in stomach cancer.
METHODS: DNA was extracted from 39 cases of surgical gastric carcinoma specimen and their correspondent normal mucosa. Seven fragments spanning the 11 exons were used to detect the mutation of p53 gene and the four exons reported to have mutations in ST7 gene were amplified by PCR and directly analyzed by DHPLC without mixing with wild-type allele.
RESULTS: In the analysis of p53 gene mutation, 9 aberrant DHPLC chromatographies were found in tumor tissues, while their normal-adjacent counterparts running in parallel showed a normal shape. Subsequent sequencing revealed nine sequence variations, 1 polymorphism and 8 mutations including 3 mutations not reported before. The mutation rate of p53 gene (21%) was consistent with that previously reported. Furthermore, no additional aberrant chromatography was found when wild-type DNA was added into the DNA of other 30 tumor samples that showed normal shapes previously. The positivity of p53 mutations was significantly higher in intestinal-type carcinomas (40%) than that in diffuse-type (8.33%) carcinomas of the stomach. No mutation of ST7 gene was found.
CONCLUSION: DHPLC is a very convenient method for the detection of somatic mutations in gastric carcinoma. The amount of wild type alleles supplied by the non-tumorous cells in gastric tumor specimens is enough to form heteroduplex with mutant alleles for DHPLC detection. ST7 gene may not be the target gene of inactivation at 7q31 site in gastric carcinoma.
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Affiliation(s)
- Chong Lu
- Laboratory of Medical Genomics, Oncology Department, the First Affiliated Clinical College, China Medical University, Shenyang, 110001, Liaoning Province, China
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24
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Rossetti S, Torra R, Coto E, Consugar M, Kubly V, Málaga S, Navarro M, El-Youssef M, Torres VE, Harris PC. A complete mutation screen of PKHD1 in autosomal-recessive polycystic kidney disease (ARPKD) pedigrees. Kidney Int 2003; 64:391-403. [PMID: 12846734 DOI: 10.1046/j.1523-1755.2003.00111.x] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND Autosomal-recessive polycystic kidney disease (ARPKD) is an important neonatal nephropathy characterized by fusiform dilation of collecting ducts, congenital hepatic fibrosis, and in some cases Caroli's disease. The ARPKD gene, PKHD1, has recently been identified. Herein we describe an effective method for PKHD1 mutation screening and the results from analysis of a novel ARPKD cohort. METHODS The coding region of PKHD1 was amplified as 79 fragments and analyzed for base pair changes by denaturing high-performance liquid chromatography (DHPLC). Forty-seven ARPKD and 14 pedigrees with congenital hepatic fibrosis and/or Caroli's disease, were screened for PKHD1 mutations. RESULTS Thirty-three different mutations were detected on 57 alleles (51.1% ARPKD, 32.1% congenital hepatic fibrosis/Caroli's disease). In the 22 pedigrees where both mutations were identified, two were homozygous for 9689delA and the remainder were compound heterozygotes; a combination of truncating, missense and splicing changes. Patients with two truncating mutations all died in the perinatal period. Two frequent truncating mutations were identified: 9689delA (9 alleles) and 5896insA (8 alleles) plus some more common missense changes; haplotype analysis indicated most were ancestral mutations. CONCLUSION DHPLC has been established as a rapid mutation screening method for ARPKD. The mutation detection rate was high in severely affected patients (85%), lower in those with moderate ARPKD (41.9%), and low, but significant, in adults with congenital hepatic fibrosis/Caroli's disease (32.1%). The prospects for gene-based diagnostics are complicated by the large gene size, marked allelic heterogeneity, and clinical diversity of the ARPKD phenotype. Identification of some common mutations, especially in specific populations, will aid mutation screening.
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Affiliation(s)
- Sandro Rossetti
- Division of Nephrology, Mayo Clinic, Rochester, Minnesota 55905, USA
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Hurtle W, Lindler L, Fan W, Shoemaker D, Henchal E, Norwood D. Detection and identification of ciprofloxacin-resistant Yersinia pestis by denaturing high-performance liquid chromatography. J Clin Microbiol 2003; 41:3273-83. [PMID: 12843075 PMCID: PMC165339 DOI: 10.1128/jcm.41.7.3273-3283.2003] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Denaturing high-performance liquid chromatography (DHPLC) has been used extensively to detect genetic variation. We used this method to detect and identify Yersinia pestis KIM5 ciprofloxacin-resistant isolates by analyzing the quinolone resistance-determining region (QRDR) of the gyrase A gene. Sequencing of the Y. pestis KIM5 strain gyrA QRDR from 55 ciprofloxacin-resistant isolates revealed five mutation types. We analyzed the gyrA QRDR by DHPLC to assess its ability to detect point mutations and to determine whether DHPLC peak profile analysis could be used as a molecular fingerprint. In addition to the five mutation types found in our ciprofloxacin-resistant isolates, several mutations in the QRDR were generated by site-directed mutagenesis and analyzed to further evaluate this method for the ability to detect QRDR mutations. Furthermore, a blind panel of 42 samples was analyzed by screening for two mutant types to evaluate the potential diagnostic value of this method. Our results showed that DHPLC is an efficient method for detecting mutations in genes that confer antibiotic resistance.
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Oefner PJ, Huber CG. A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 782:27-55. [PMID: 12457994 DOI: 10.1016/s1570-0232(02)00700-6] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The introduction of alkylated, nonporous poly-(styrene-divinylbenzene) microparticles in 1992 enabled the subsequent development of denaturing HPLC that has emerged as the most sensitive screening method for mutations to date. Denaturing HPLC has provided unprecedented insight into human origins and prehistoric migrations, accelerated the cloning of genes involved in mono- and polygenic traits, and facilitated the mutational analysis of more than a hundred candidate genes of human disease. A significant step toward increased sample-throughput and information content was accomplished by the recent introduction of monolithic poly(styrene-divinylbenzene) capillary columns. They have enabled the construction of capillary arrays amenable to multiplex analysis of fluorescent dye-labeled nucleic acids by laser-induced fluorescence detection. Hyphenation of denaturing HPLC with electrospray ionization mass spectrometry, on the other hand, has allowed the direct elucidation of the chemical nature of DNA variation and determination of phase of multiple alleles on a chromosome.
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Affiliation(s)
- Peter J Oefner
- Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto 94304, USA.
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27
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Weirich G, Cabras AD, Serra S, Coni PP, Nurchi AM, Faa G, Höfler H. Rapid identification of Wilson's disease carriers by denaturing high-performance liquid chromatography. Prev Med 2002; 35:278-84. [PMID: 12202071 DOI: 10.1006/pmed.2002.1069] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
BACKGROUND Wilson's disease is an autosomal recessive disorder characterized by decreased biliary copper excretion and reduced copper incorporation into ceruloplasmin. The disease gene ATP7B maps to chromosome 13q14.3, contains 21 exons, and encodes a copper-transporting P-type ATPase. ATP7B mutations are scattered over the entire gene, and scanning methods to detect mutation carriers are in demand. We have tested the usefulness of denaturing high-performance liquid chromatography for mutation detection in Wilson's disease. METHODS Genomic DNA from five Sardinian Wilson's disease families (32 individuals, 8 patients) was subjected to polymerase chain reactions for ATP7B exons 2-21 and the 5' untranslated region. PCR products were analyzed by chromatography and by direct sequencing. RESULTS Three disease-causing mutations and seven sequence variants were detected by chromatography. Five patients were homozygotes for -441/-427del, and three were compound heterozygotes for V1146M plus 1512-13insT (N505X) and for -441/-427del plus V1146M, respectively. Eighteen unaffected individuals were mutation carriers. Sequence variants comprised V290V, A406S, L456V, R832K, A1140V, the novel K952R, and T991T. The novel intronic IVS18+6c>t change escaped detection by chromatography. CONCLUSIONS Denaturing high-performance liquid chromatography is a dependable tool for ATP7B screening that is superior to traditional haplotyping. This method allows for fast, sensitive, and specific mutation detection and identification of carriers in Wilson's disease families.
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Affiliation(s)
- Gregor Weirich
- Institute of Pathology, Technische Universität München, Trogerstrasse 18, D-81675 Munich, Germany.
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Liu MR, Pan KF, Li ZF, Wang Y, Deng DJ, Zhang L, Lu YY. Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography. World J Gastroenterol 2002; 8:426-30. [PMID: 12046063 PMCID: PMC4656414 DOI: 10.3748/wjg.v8.i3.426] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA).
METHODS: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen.
RESULTS: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with Apa I. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5% mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e.g., Pfu) in eliminating the false positive produced by Taq DNA polymerases.
CONCLUSION: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.
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Affiliation(s)
- Man-Ran Liu
- Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, Peking University, Western District, Beijing 100034, China
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Rossetti S, Chauveau D, Walker D, Saggar-Malik A, Winearls CG, Torres VE, Harris PC. A complete mutation screen of the ADPKD genes by DHPLC. Kidney Int 2002; 61:1588-99. [PMID: 11967008 DOI: 10.1046/j.1523-1755.2002.00326.x] [Citation(s) in RCA: 120] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND Genetic analysis is a useful diagnostic tool in autosomal dominant polycystic kidney disease (ADPKD), especially when imaging results are equivocal. However, molecular diagnostics by direct mutation screening has proved difficult in this disorder due to genetic and allelic heterogeneity and complexity of the major locus, PKD1. METHODS A protocol was developed to specifically amplify the exons of PKD1 and PKD2 from genomic DNA as 150 to 450 bp amplicons. These fragments were analyzed by the technique of denaturing high-performance liquid chromatography (DHPLC) using a Wave Fragment Analysis System (Transgenomics) to detect base-pair changes throughout both genes. DHPLC-detected changes were characterized by sequencing. RESULTS Cost effective and sensitive mutation screening of the entire coding regions of PKD1 and PKD2 by DHPLC was optimized. All base-pair mutations to these genes that we previously characterized were detected as an altered DHPLC profile. To assess this method for routine diagnostic use, samples from a cohort of 45 genetically uncharacterized ADPKD patients were analyzed. Twenty-nine definite mutations were detected, 26 PKD1, 3 PKD2 and a further five possible missense mutations were characterized leading to a maximal detection rate of 76%. A high level of polymorphism of PKD1 also was detected, with 71 different changes defined. The reproducibility of the DHPLC profile enabled the recognition of many common polymorphisms without the necessity for re-sequencing. CONCLUSIONS DHPLC has been demonstrated to be an efficient and effective means for gene-based molecular diagnosis of ADPKD. Differentiating missense mutations and polymorphisms remains a challenge, but family-based segregation analysis is helpful.
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Affiliation(s)
- Sandro Rossetti
- Division of Nephrology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA
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Colosimo A, Guida V, De Luca A, Cappabianca MP, Bianco I, Palka G, Dallapiccola B. Reliability of DHPLC in mutational screening of beta-globin (HBB) alleles. Hum Mutat 2002; 19:287-95. [PMID: 11857746 DOI: 10.1002/humu.10046] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The inherited disorders of hemoglobin represent the most common Mendelian disease worldwide, with a higher prevalence among Mediterraneans, Asians, Africans, and Indians. Altered beta-globin sequences, causing either hemoglobinopathies or beta-thalassemia syndromes, are due to more than 200 different mutations in the beta-globin gene. Prevention programs based on postnatal and prenatal molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. We have developed a rapid and highly specific mutation screening test based on the denaturing high-performance liquid chromatography (DHPLC) system. The sensitivity and specificity of the method were tested on the full genomic region of the beta-globin gene in 30 normal Italian subjects and 40 heterozygous carriers in which 25 different beta-globin mutations had been previously characterized by multiplex-ARMS technique. The results showed DHPLC to be 100% sensitive and specific. All the 25 sequence alterations and two previously undetected polymorphisms were precisely identified with neither false positive nor false negative results. In addition, 12 compound heterozygous and four homozygous patients were successfully subjected to DHPLC. Overall, the method was able to rapidly identify the most common beta-globin mutations, accounting for more than 97% of beta-globin alleles in the Italian population. Compared to classical approaches of mutation screening, this method allows a rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.
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