Molecular Test For Inherited Mutations In Breast Cancer
Mark Lim
Ambergen, Inc
Grant 5R44CA110403-04 from National Cancer Institute, IRG: ZRG1
Abstract: Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are sporadic (e.g. not inherited), approximately 10% are due to inherited mutations in the BRCA1 and BRCA2 genes leading to a lifetime risk of 85% for breast cancer and 40-65% for ovarian cancer. Importantly, recent progress in understanding the molecular basis for inherited breast and ovarian cancer has led to the development of candidate drugs (PARP inhibitors) which for the first time offer the hope of preventing and treating cancer in women carrying defective BRCA1/2 genes. There is also increasing evidence that pre-surgical genetic screening of breast cancer patients helps assist in deciding on therapeutic options. Therefore, it is becoming increasingly important to be able to identify carriers of BRCA1/2 mutations. However, current breast cancer susceptibility gene testing which involves DNA sequencing is prohibitively expensive in many cases. The objective of this project is to develop a cost-effective technology to screen for mutations in the BRCA1/2 genes. Two related methods have been successfully tested in Phase I, both based on in vitro expression of peptides from overlapping segments of PCR amplified BRCA1/2 genomic DNA and mRNA. One approach utilizes an ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations which constitute 95% of all deleterious mutations in BRCA1/2. A second approach, utilizes mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) to scan for all possible mutations, including missense mutations. In both cases, studies on normal and mutant bearing cell-lines and a limited number of patient samples reveals the tests are highly effective scanning techniques with a sensitivity approaching DNA sequencing. During Phase II, we propose to further develop and extensively evaluate these novel BRCA1/2 assays. The ELISA-PTT chain-truncation assay developed in Phase I will be improved to cover the entire BRCA1/2 coding region using single-step PCR reactions. The test will utilize a combination of DNA and mRNA extracted from blood. The MASSIVE-PRO assay will also be extended to scan for both chain truncations and missense mutations in both genes. Advanced technology based on several innovations, which can significantly reduce the number of PCR, and cell-free reactions will be evaluated. The new assay will be extensively evaluated in collaboration with Dr. Jessica K. Booker, Associate Director of the Molecular Diagnostics Laboratory at the University of North Carolina School Of Medicine (UNCSM) using a repository of 250 pre-validated genomic DNA and mRNA samples from patients screened for hereditary breast cancer. Clinical a-testing will be performed during the third year in both Dr. Booker´s laboratory and at Quest Diagnostics, Inc. Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are not inherited, over 20,000 cases per year in the U.S. are due to inherited mutations in two specific genes leading to a lifetime risk of 85% for breast cancer and 40- 65% for ovarian cancer. Importantly, a new generation of drugs are being developed which target hereditary breasted cancer. However, current breast cancer susceptibility gene testing is often prohibitively expensive for patients and insurance companies. The objective of this project is to develop a new, cost-effective technology to screen for mutations indicative of hereditary breast cancer. A new approach to detect these breast cancer mutations developed by AmberGen will be extensively tested during this project. This test promises to be rapid, highly sensitive, low-cost and easy to perform in a typical clinical laboratory setting. If successful, the test will enable more extensive screening and consequently increased surveillance and early treatment for hereditary breast cancer
Project start date: 2004-09-01
Project end date: 2009-07-31
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Molecular Test For Inherited Mutations In Breast Cancer
Mark Lim
Ambergen, Inc Watertown, Ma 02472
Grant 5R44CA110403-03 from National Cancer Institute, IRG: ZRG1
Abstract: Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are sporadic (e.g. not inherited), approximately 10% are due to inherited mutations in the BRCA1 and BRCA2 genes leading to a lifetime risk of 85% for breast cancer and 40-65% for ovarian cancer. Importantly, recent progress in understanding the molecular basis for inherited breast and ovarian cancer has led to the development of candidate drugs (PARP inhibitors) which for the first time offer the hope of preventing and treating cancer in women carrying defective BRCA1/2 genes. There is also increasing evidence that pre-surgical genetic screening of breast cancer patients helps assist in deciding on therapeutic options. Therefore, it is becoming increasingly important to be able to identify carriers of BRCA1/2 mutations. However, current breast cancer susceptibility gene testing which involves DNA sequencing is prohibitively expensive in many cases. The objective of this project is to develop a cost-effective technology to screen for mutations in the BRCA1/2 genes. Two related methods have been successfully tested in Phase I, both based on in vitro expression of peptides from overlapping segments of PCR amplified BRCA1/2 genomic DNA and mRNA. One approach utilizes an ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations which constitute 95% of all deleterious mutations in BRCA1/2. A second approach, utilizes mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) to scan for all possible mutations, including missense mutations. In both cases, studies on normal and mutant bearing cell-lines and a limited number of patient samples reveals the tests are highly effective scanning techniques with a sensitivity approaching DNA sequencing. During Phase II, we propose to further develop and extensively evaluate these novel BRCA1/2 assays. The ELISA-PTT chain-truncation assay developed in Phase I will be improved to cover the entire BRCA1/2 coding region using single-step PCR reactions. The test will utilize a combination of DNA and mRNA extracted from blood. The MASSIVE-PRO assay will also be extended to scan for both chain truncations and missense mutations in both genes. Advanced technology based on several innovations, which can significantly reduce the number of PCR, and cell-free reactions will be evaluated. The new assay will be extensively evaluated in collaboration with Dr. Jessica K. Booker, Associate Director of the Molecular Diagnostics Laboratory at the University of North Carolina School Of Medicine (UNCSM) using a repository of 250 pre-validated genomic DNA and mRNA samples from patients screened for hereditary breast cancer. Clinical a-testing will be performed during the third year in both Dr. Booker s laboratory and at Quest Diagnostics, Inc. Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are not inherited, over 20,000 cases per year in the U.S. are due to inherited mutations in two specific genes leading to a lifetime risk of 85% for breast cancer and 40- 65% for ovarian cancer. Importantly, a new generation of drugs are being developed which target hereditary breasted cancer. However, current breast cancer susceptibility gene testing is often prohibitively expensive for patients and insurance companies. The objective of this project is to develop a new, cost-effective technology to screen for mutations indicative of hereditary breast cancer. A new approach to detect these breast cancer mutations developed by AmberGen will be extensively tested during this project. This test promises to be rapid, highly sensitive, low-cost and easy to perform in a typical clinical laboratory setting. If successful, the test will enable more extensive screening and consequently increased surveillance and early treatment for hereditary breast cancer.
Project start date: 2004-09-01
Project end date: 2009-07-31
5R44CA110403-03 (2007): $463209
Grants awarded to Mark Lim
High Sensitivity Molecular Test For Colorectal Cancer
Mark Lim
Ambergen, Inc Watertown, Ma 02472
Grant 1R43CA106844-01A1 from National Cancer Institute, IRG: ZRG1
Abstract: There exists an urgent need to develop an effective, non-invasive method of detecting colorectal cancer (CRC), the second leading cause of cancer deaths in the U.S. Such non-invasive testing, if instituted for a large segment of the population, could result in a dramatic reduction in the approximately 55,000 annual deaths caused by CRC. However, current methods for early detection of CRC, which include the fecal occult-blood test (FOBT) and endoscopic colorectal examination (colonoscopy), either have nominal effectiveness due to low sensitivity (FOBT) or very low compliance due to rigorous preparative requirements (colonoscopy). The principal objective of this project is to develop a cost-effective and high throughput method for detection of molecular markers of CRC from fecal DNA. CRC assays on fecal DNA must also be highly sensitive, since mutations characteristic of CRC are likely to be present in concentrations relative to WT of less than 1%. Chain truncating mutations in the APC gene have the highest genetic correlation (approximately 80%) known for CRC. However, full-sequencing of the APC gene is prohibitively expensive due to its large size (8.5 Kb) and lacks sufficient sensitivity, while targeting the known mutations would require over 1000 probes and still miss significant de novo mutations. As an alternative, a newly developed ELISA-based protein truncation test (ELISA-PTT) will be evaluated. In contrast to conventional protein truncation tests, ELISA-PTT eliminates the need for electrophoresis and radioactivity, making it cost-effective and suitable for high throughput applications. ELISA-PTT will be combined with digital PCR and subractive techniques in order to detect chain-truncating mutants at concentrations as low as 0.4% present in DNA extracted from stool. Preliminary studies demonstrate AmberGen s ability to routinely isolate fecal DNA from small stool samples (>2mg). During Phase I, this new approach will be further evaluated using CRC repositories of stool samples as well as new samples obtained from pre-operative CRC patients in collaboration with Dr. Paul C. Schroy, Director of Clinical Research for the Section of Gastroenterology at Boston Medical Center. Dr. Tim Heeren, Professor of Biostatistics at the Boston University School of Public Health, will assist in statistical analysis of data. The results obtained with these new technologies will be validated using full-sequencing of DNA extracted from surgically removed tumors tissue. During Phase II, an optimized non-invasive assay system will be evaluated for CRC population screening.
Keywords: colorectal neoplasm, diagnosis design /evaluation, enzyme linked immunosorbent assay, gene mutation, high throughput technology, molecular oncology, neoplasm /cancer diagnosis, noninvasive diagnosis, polymerase chain reaction, tumor suppressor gene, DNA, adenomatous polyp, cost effectiveness, biotechnology, clinical research, feces analysis, human subject, human tissue
Project start date: 2005-05-01
Project end date: 2007-04-30
1R43CA106844-01A1 (2005): $187250
Cell-Free Protein Labeling For Drug Discovery
Mark Lim
Ambergen, Inc Watertown, Ma 02472
Grant 5R44GM063369-03 from National Institute Of General Medical Sciences, IRG: ZRG1
Abstract: The human genome has opened a new chapter for drug discovery. In contrast to conventional drug discovery methods, which screen a drug library against a single protein target, the entire protein complement of the genome, i.e. the proteome, is now available for screening. A proteomic screen would greatly accelerate the drug discovery process by allowing potential interactions between a lead drug compound and an entire proteome to be determined. However, such drug-proteomic screens would be difficult to perform using conventional technology because of the need to rapidly convert genome to proteome, the requirement of engineering of proteins with specialized detection and affinity tags and the need for a platform that has the high throughput necessary to examine thousands of drug-protein interactions per hour. During Phase I, we have developed a new approach for drug discovery based on recent progress in the areas of in vitro protein expression, tRNA mediated protein engineering (TRAMPE) and photocleavable (PC) linkers such as PC-biotin. This technology allows the direct expression of individual proteins from genes or entire proteomes from genomes. A key to this technology is the incorporation of specially designed markers and affinity tags into proteins during their in vitro expression, thus facilitating their rapid isolation and detection. A central approach, termed PC-SNAG, allows proteins to be rapidly isolated from complex in vitro expression mixtures on beads and then photoreleased in pure form into solution or transferred directly onto the surface of a microarray substrate. During Phase II, proof-of-concept will be established for this new technology by creating a 321 member kinase library of in vitro expressed proteins (LIVE-PRO TM) that will be formatted on photoreleasable beads. In one assay, LIVE-PRO will be simultaneously screened to detect potential interactions with known kinase inhibitors. In a second assay, the effects of specific kinase inhibitors on the activity of each member of the LIVE-PRO will be simultaneously measured. This work will be performed in collaboration with Invitrogen, whose Gateway TM recombination technology and human Ultimate TM ORF Clones will allow creation of libraries of in vitro expressible plasmids. Dr. Mike Pavia, the former Chief Technology Officer at Millennium Pharmaceuticals and currently Entrepreneur-in-Residence at Oxford Bioscience will serve as a consultant.
Keywords: cell free system, drug discovery /isolation, enzyme inhibitor, peptide library, protein kinase, proteomics, technology /technique development, drug interaction, drug screening /evaluation, gene expression, genetic library, plasmid, transfer RNA, bioengineering /biomedical engineering, high throughput technology, microarray technology
Project start date: 2001-05-01
Project end date: 2007-05-31
5R44GM063369-03 (2005): $374969
ADVANCED TECHNOLOGY FOR ASSAYING CANCER-DRUG RESISTANCE
Mark Lim
Ambergen, Inc
Grant 2R44CA114126-02 from National Cancer Institute, IRG: ZCA1
Abstract: Significant advances have been made toward the development of a new generation of molecularly targeted cancer drugs, many of which are only now emerging from the pipeline. This project aims to develop a new, highly sensitive technology for detecting drug-resistance mutations in proteins which preexist prior to treatment or are acquired due to the selective pressure exerted by treatment with molecularly targeted anti-cancer drugs (ACD). This problem is exemplified by drug resistance developed in patients treated for chronic myeloid leukemia (CML) with the small molecule drug Imatinib (Gleevec/Glivec/STI571). It is well documented that this resistance arises from mutations in BCR-ABL tyrosine kinase, the target for Imatinib. Drug resistance also occurs in both Philadelphia chromosome positive (Ph+) acute lymphatic leukemia (Ph+ ALL) and gastrointestinal stromal tumors (GIST) patients who are treated with Imatinib. Several problems must be overcome in order to effectively detect and characterize drug resistance mutations against anti-cancer drugs i) The spectrum of mutations can be very diverse, occurring outside the drug binding pocket. In the case of the BCR-ABL kinase, mutations appear throughout the kinase domain, such as the P-loop (ATP binding) and A-loop (regulatory region). This can necessitate DNA sequencing of the entire gene or specific portions in order to detect the occurrence of both known and uncharacterized mutations. However, DNA sequencing is expensive when incorporated into a commercial assay and has limited sensitivity (>20%). ii) It is possible to isolate the drug target proteins or fragments to perform functional and/or structural analysis. However, such analyses are hindered by difficulties in assaying targets in crude biological mixtures and in isolating target proteins in a pure form. In this project these limitations are overcome by using novel technology developed by AmberGen for isolating highly purified cell-free expressed polypeptide fragments of the drug-targeted protein(s), and detecting characteristic drug resistance mutations using a matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) scanning technique. The new approach, termed drug resistance assay for mutations against anti-cancer drugs (DRAMA-ACD) has the advantage that it allows low-cost scanning for mutations (even those previously undiscovered) with high sensitivity and high throughput. Achievement of key milestones during Phase I included i) the demonstration that DRAMA-ACD could detect mutant BCR-ABL tyrosine kinase at a sensitivity of at least 5%; ii) the successful demonstration of PC-SNAG, a method of capturing and photoreleasing cell-free expressed fragments of the BCR-ABL tyrosine kinase with significantly reduced levels of non-specific contamination; iii) the demonstration that all steps in DRAMA-ACD can be multiplexed using bead-sorted libraries of in vitro expressed proteins (BS-LIVE-PRO); iv) Development of a process known as PC-PRINT which enables beads containing the target peptides to be transferred directly to a MALDI-MS target for direct analysis. During Phase II, we will continue to focus on the development and application of DRAMA-ACD to detect mutations in the BCR-ABL tyrosine kinase. An important milestone will be a demonstration of the ability to detect these mutations in CML patients with a sensitivity of 1%. The research will be carried out in collaboration with Dr. Adam Lerner, Associate Professor of Medicine and Pathology, a leading expert in the area of hematologic malignancies, who will provide us with samples for analysis from CML and Ph+ ALL patients undergoing Imatinib treatment. All results will be statistically analyzed in collaboration with Prof. Jose Dupuis, Associate Professor of Biostatistics, and Boston University School of Public Health. We will also maintain a close contact during Phase II with two leading diagnostic companies, LabCorp and Genzyme Genetics, who have expressed an interest in the ultimate commercialization of the DRAMA-ACD approach
Project start date: 2005-07-07
Project end date: 2011-06-30
1R43CA114126-01 (2005): $183926
Photocleavable ICAT Reagents For Quantitative Proteomics
Mark Lim
Ambergen, Inc Watertown, Ma 02472
Grant 5R44GM070325-03 from National Institute Of General Medical Sciences, IRG: ZRG1
Abstract: Protein expression profiling is becoming one of the most attractive tools for monitoring the course of neoplastic disease as well as identifying diagnostic biomarkers associated with the disease. While several proteomic tools have been extensively investigated for this purpose, including two-dimensional gel electrophoresis and mass spectrometry, quantitative proteomics is still difficult to achieve due to the limited accuracy and inability of these methods to survey the entire proteome. The goal of this project is to overcome these limitations by developing a new approach to quantitative proteomics based on the use of PhotoCleavable Isotope Coded Affinity Tags (PC-ICAT) along with mass spectrometry. PC-ICAT reagents are composed of four distinct elements a biotin affinity tag, a photodeavable linker which exhibits rapid and efficient photocleavage, a variable mass linker and a chemically reactive group. The light and heavy versions of the PC-ICAT reagents are used to tag two different samples, for example extracts from normal and disease related tissues. After digestion, capture and photorelease, the relative abundance of the tagged peptides in each sample is measured using LC-MS. During Phase I of this project, a sulfhydryl reactive PC-ICAT maleimide reagent has been synthesized and extensively evaluated using both model peptides and proteins. Significantly, the performance of PC-ICAT exhibits superior performance compared with the conventional (CAT reagent. During Phase II, we will extend the usefulness of PC-ICAT in proteomic research and clinical applications by 1) synthesizing and evaluating an amine reactive version of the PC-ICAT reagent, 2) evaluating a solid supported PC-ICAT reagent and 3) exploring the use of PC-ICAT reagents for protein expression profiling and biomarker discovery. For this purpose, we will apply PC-ICAT reagents and LC-MS to study cell lysates derived from normal and malignant human prostate cell lines. Dr. Cathy Costello, an expert in the area of mass spectrometry and proteomics will serve as a consultant during Phase II of this project.
Keywords: protein, proteomics, amine, avidin, base, binding protein, biomarker, biotin, buffer, cell, cell line, clinical research, conditioning, digestion, electrophoresis, element, family, gel, human, identity, lighting, maleimide, mass spectrometry, model, neoplastic cell, peptide, performance, prostate, prostate neoplasm, sectioning, spectrometry, tissue
Project start date: 2004-01-01
Project end date: 2009-03-31
5R44GM070325-03 (2007): $374500
2R44GM070325-02 (2006): $374500
Novel Proteomic Arrays Of In Vitro Expressed Proteins For Autoimmune Disease
Mark Lim
Ambergen, Inc
watertown, Ma 02472
Grant 5R44AI052525-04 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1
Abstract: Progress in sequencing the human genome has led to a new goal - expressing and characterizing the human proteome. A promising area where proteomics can have a major impact is autoimmune disease. Proteome-wide screening against sera from patients with particular autoimmune diseases can enable the discovery of new autoantigens, the development of microarray-based diagnostic assays, and effective treatments for autoimmune disease. During Phase I, we developed a novel, low-cost, high throughput approach for proteomics based on Bead Sorted Libraries of In Vitro Expressed Proteins (BS-LIVE- PRO). These protein libraries can be expressed inexpensively in a single cell-free translation reaction using a Bead Sorted Library of In Vitro Expressible DNA (BS-LIVE-DNA) as a template. Additional novel technologies developed during Phase I which augment this approach include i) solid-phase PCR to produce BS-LIVE-DNA from cDNA libraries, ii) a method (PC-PRINT) to rapidly phototransfer the protein on each bead in the BS-LIVE-PRO onto discrete spots on a microarray surface using proprietary photocleavable linkers, and iii) methods to decode the randomly arrayed spots generated by PC-PRINT using photo-transferable DNA or mass-tags (PC-CODE). We propose to extensively optimize and evaluate this new technology during Phase II with the aim of commercializing BS-LIVE-PRO. We will apply BS-LIVEPRO to autoimmune diseases in collaboration with Dr. Donald Bloch at the Massachusetts General Hospital, a leading expert on autoantigen discovery and primary biliary cirrhosis. Similar studies will be carried out on other vasculitis autoimmune diseases in association with Dr. Peter Merkel, Director of the Vasculitis Research Consortium. Proteome-wide screening against sera from patients with particular autoimmune diseases can lead to the discovery of new autoantigens, the development of diagnostic assays to identify autoimmune disease and, ultimately, improved treatments. We have developed a novel, low-cost, high throughput approach for proteomics based on Bead Sorted Libraries of In Vitro Expressed Proteins (BS-LIVE-PRO). We propose to extensively optimize and evaluate this new technology during Phase II with the aim of commercializing diagnostic assays for autoimmune diseases
Keywords: autoantigen, base, library, protein, proteomics DNA, antibody, antigen, autoantibody, biology, cell, diagnosis, drug discovery /isolation, fluorescence, genome, health /scientific organization, hospital, human, lead, primary biliary cirrhosis, school, sectioning, serum, university, vasculitis
Project start date: 2002-09-01
Project end date: 2010-03-31
2R44AI052525-03A1 (2007): $657583
Molecular Test For Inherited Mutations In Breast Cancer
Mark Lim
Ambergen, Inc
Grant 5R44CA110403-04 from National Cancer Institute, IRG: ZRG1
Abstract: Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are sporadic (e.g. not inherited), approximately 10% are due to inherited mutations in the BRCA1 and BRCA2 genes leading to a lifetime risk of 85% for breast cancer and 40-65% for ovarian cancer. Importantly, recent progress in understanding the molecular basis for inherited breast and ovarian cancer has led to the development of candidate drugs (PARP inhibitors) which for the first time offer the hope of preventing and treating cancer in women carrying defective BRCA1/2 genes. There is also increasing evidence that pre-surgical genetic screening of breast cancer patients helps assist in deciding on therapeutic options. Therefore, it is becoming increasingly important to be able to identify carriers of BRCA1/2 mutations. However, current breast cancer susceptibility gene testing which involves DNA sequencing is prohibitively expensive in many cases. The objective of this project is to develop a cost-effective technology to screen for mutations in the BRCA1/2 genes. Two related methods have been successfully tested in Phase I, both based on in vitro expression of peptides from overlapping segments of PCR amplified BRCA1/2 genomic DNA and mRNA. One approach utilizes an ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations which constitute 95% of all deleterious mutations in BRCA1/2. A second approach, utilizes mass spectrometry of in vitro expressed proteins (MASSIVE-PRO) to scan for all possible mutations, including missense mutations. In both cases, studies on normal and mutant bearing cell-lines and a limited number of patient samples reveals the tests are highly effective scanning techniques with a sensitivity approaching DNA sequencing. During Phase II, we propose to further develop and extensively evaluate these novel BRCA1/2 assays. The ELISA-PTT chain-truncation assay developed in Phase I will be improved to cover the entire BRCA1/2 coding region using single-step PCR reactions. The test will utilize a combination of DNA and mRNA extracted from blood. The MASSIVE-PRO assay will also be extended to scan for both chain truncations and missense mutations in both genes. Advanced technology based on several innovations, which can significantly reduce the number of PCR, and cell-free reactions will be evaluated. The new assay will be extensively evaluated in collaboration with Dr. Jessica K. Booker, Associate Director of the Molecular Diagnostics Laboratory at the University of North Carolina School Of Medicine (UNCSM) using a repository of 250 pre-validated genomic DNA and mRNA samples from patients screened for hereditary breast cancer. Clinical a-testing will be performed during the third year in both Dr. Booker´s laboratory and at Quest Diagnostics, Inc. Breast cancer is the most common malignancy which occurs in women and the second leading cause of cancer death. While the majority of breast cancers are not inherited, over 20,000 cases per year in the U.S. are due to inherited mutations in two specific genes leading to a lifetime risk of 85% for breast cancer and 40- 65% for ovarian cancer. Importantly, a new generation of drugs are being developed which target hereditary breasted cancer. However, current breast cancer susceptibility gene testing is often prohibitively expensive for patients and insurance companies. The objective of this project is to develop a new, cost-effective technology to screen for mutations indicative of hereditary breast cancer. A new approach to detect these breast cancer mutations developed by AmberGen will be extensively tested during this project. This test promises to be rapid, highly sensitive, low-cost and easy to perform in a typical clinical laboratory setting. If successful, the test will enable more extensive screening and consequently increased surveillance and early treatment for hereditary breast cancer
Project start date: 2004-09-01
Project end date: 2009-07-31
2R44CA110403-02 (2006): $463209
Advanced Molecular Diagnostic Test For Neurofibromatosis
Mark Lim
Ambergen, Inc
Grant 5R44NS053110-03 from National Institute Of Neurological Disorders And Stroke, IRG: ZRG1
Abstract: Advanced Molecular Diagnostic Test for Neurofibromatosis Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene. In children, NF1 causes learning disabilities, coordination problems and benign tumors later in life that grow on nerve tissue. Currently more than 100,000 Americans suffer from NF1 with the disease having one of the highest prevalence rates for a genetic disorder (~1 out of 4,000 births). Progress has recently been made in identifying possible drugs for this disease including the proposed use of statins to alleviate learning disabilities in children with NF1. There is a critical need for a cost-effective test to screen for mutations in the NF1 gene. However, a significant problem is the wide variety of NF1 mutations which have been found to occur. Most inactivating mutations (>80%) are chain truncating and distributed throughout the gene. The preferred method to detect such mutations is the protein truncation test (PTT). However, because of the many limitations of conventional PTT, it is not practical for routine clinical use. Instead, current diagnosis is normally based on established non-genetic clinical criteria. Significant progress was made during Phase I in overcoming these limitations by developing a cost- effective alternative to conventional PTT. One approach utilizes an ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations. In contrast to conventional PTT, ELISA-PTT eliminates the need for electrophoresis and radioactivity. A second and complementary approach, based on mass spectrometric analysis of in vitro expressed proteins (MASSIVE-PRO), is able to additionally scan for missense mutations by precisely measuring the mass shift from single amino acid substitutions. Studies performed using these improved assays on normal and mutant cDNAs as well a limited number of NF1 patient samples revealed that these tests are highly effective with sensitivities comparable to and in some cases superior to standard DNA sequencing. During Phase II, we will further develop and extensively evaluate these novel NF1 diagnostic assays. The ELISA-PTT will be improved to allow scanning of the entire NF1 coding region using mRNA isolated from patient blood and a single-step PCR protocol. The MASSIVE-PRO assay will also be improved so that it can provide specific sequence information about mutations in the NF1 gene. Advanced technologies based on several innovations which can significantly reduce the number of PCR and cell-free translation reactions will also be evaluated. These assays, once optimized, will be extensively evaluated in collaboration with Dr. Ludwine Messiaen, Director of Genomics at the University of Alabama and a world leader in NF1 testing using a repository of validated genomic DNA and mRNA samples from NF1 patients. Clinical beta-testing will be performed during the third year in Dr. Messiaen´s laboratory. In addition, Quest Diagnostics will work closely with AmberGen in order to assure these NF1 tests are properly designed for the requirements of the clinical laboratory environment. Advanced Molecular Diagnostic Test for Neurofibromatosis NARRATIVE Currently more than 100,000 Americans suffer from neurofibromatosis type 1 (NF1) which is among the most prevalent genetic disorders (~1 out of 4,000 births). An important goal is to develop a cost- effective test to screen for mutations in the NF1 gene. Significant progress was made during Phase I in developing a cost-effective alternative to conventional genetic tests. During Phase II, we propose to further develop and extensively evaluate these novel NF1 mutation assays in collaboration with Dr. Ludwine Messiaen, Director of Genomics at the University of Alabama and a world leader in NF1 testing using a repository of validated genomic DNA and mRNA samples. If successful, the test will enable more extensive screening and consequently increased surveillance and better management of NF1
Keywords: diagnostic test, gene, gene mutation, neurofibromatosis DNA, RNA, ing, acid, attention, autoradiography, base, birth, blood, cell, cell line, children, cognition disorder, diagnosis, electrophoresis, environment, family, fibroma, gel, genetic disorder, genetics, human, learning, learning disorder, mass spectrometry, memory, molecular weight, mutant, neoplasm /cancer, neoplastic growth, nerve, nucleic acid sequence, point mutation, protein, sectioning, skin, tissue, university
Project start date: 2005-09-15
Project end date: 2010-06-30
2R44NS053110-02 (2007): $383348
Related Publications
Photocleavage-based affinity purification and printing of cell-free expressed proteins: Application to proteome microarrays. Anal Biochem. 2008 Aug 12. [Epub ahead of print] PMID: 18762158 Quantitative and label-free technique for measuring protease activity and inhibition using a microfluidic cantilever array. Nano Lett. 2008 Sep; 8( 9): 2968-74. Epub 2008 Aug 23. PMID: 18720973 
Cell-free protein synthesis systems: biotechnological applications. Biotechnol Genet Eng Rev. 2006; 22: 151-69. Review. No abstract available. PMID: 18476330 Using specificity to strategically target proteases. Bioorg Med Chem. 2008 Mar 30. [Epub ahead of print] PMID: 18434168 Amine nitrosation via NO reduction of the polyamine copper(II) complex Cu(DAC)2+. Inorg Chem. 2007 Oct 29; 46( 22): 9323-31. Epub 2007 Sep 27. PMID: 17900109 Probing shapes of bichromophoric metal-organic complexes using ion mobility mass spectrometry. J Am Chem Soc. 2005 Dec 28; 127( 51): 18222-8. PMID: 16366575 [PubMed] The preparation of anaerobic nitric oxide solutions for the study of heme model systems in aqueous and nonaqueous media: some consequences of NO x impurities. Methods Enzymol. 2005; 396: 3-17. PMID: 16291216 Mechanisms of reductive nitrosylation in iron and copper models relevant to biological systems. Chem Rev. 2005 Jun; 105( 6): 2439-55. Review. No abstract available. PMID: 15941218 Further evidence supporting an inner sphere mechanism in the NO reduction of the copper(II) complex Cu(dmp)2(2+) (dmp=2,9-dimethyl-1,10-phenanthroline). Nitric Oxide. 2005 Jun; 12( 4): 244-51. PMID: 15917217 Reactions of nitrogen oxides with heme models. Spectral and kinetic study of nitric oxide reactions with solid and solute Fe(III)(TPP)(NO3). J Am Chem Soc. 2005 May 4; 127( 17): 6216-24. PMID: 15853326 A cyclic tetra-nuclear dinitrosyl iron complex [Fe(NO)2(imidazolate)]4: synthesis, structure and stability. Chem Commun (Camb). 2005 Jan 28;( 4): 477-9. Epub 2004 Dec 14. PMID: 15654375 NO and NOx interactions with group 8 metalloporphyrins. J Inorg Biochem. 2005 Jan; 99( 1): 151-65. Review. PMID: 15598499 Intramolecular reductive nitrosylation: reaction of nitric oxide and a copper(II) complex of a cyclam derivative with pendant luminescent chromophores. J Am Chem Soc. 2004 Jun 2; 126( 21): 6564-5. PMID: 15161279 A high-throughput nonisotopic protein truncation test. Nat Biotechnol. 2003 Feb; 21( 2): 194-7. Epub 2003 Jan 13. Erratum in: Nat Biotechnol. 2003 Sep;21(9):1098. PMID: 12524552 Reactions of nitrogen oxides with heme models. Characterization of NO and NO2 dissociation from Fe(TPP)(NO2)(NO) by flash photolysis and rapid dilution techniques: Fe(TPP)(NO2) as an unstable intermediate. J Am Chem Soc. 2002 Aug 21; 124( 33): 9737-43. PMID: 12175231 Kinetics of the oxidation of triphenylphosphine by nitric oxide. Inorg Chem. 2002 Feb 25; 41( 4): 1026-8. No abstract available. PMID: 11849111 [PubMed]