Protein-coding genetic variants that strongly affect disease risk can yield relevant clues to disease pathogenesis. Here we report exome-sequencing analyses of 20,791 individuals with type 2 diabetes (T2D) and 24,440 non-diabetic control participants from 5 ancestries. We identify gene-level associations of rare variants (with minor allele frequencies of less than 0.5%) in 4 genes at exome-wide significance, including a series of more than 30 SLC30A8 alleles that conveys protection against T2D, and in 12 gene sets, including those corresponding to T2D drug targets (P = 6.1 × 10−3) and candidate genes from knockout mice (P = 5.2 × 10−3). Within our study, the strongest T2D gene-level signals for rare variants explain at most 25% of the heritability of the strongest common single-variant signals, and the gene-level effect sizes of the rare variants that we observed in established T2D drug targets will require 75,000–185,000 sequenced cases to achieve exome-wide significance. We propose a method to interpret these modest rare-variant associations and to incorporate these associations into future target or gene prioritization efforts. Exome-sequencing analyses of a large cohort of patients with type 2 diabetes and control individuals without diabetes from five ancestries are used to identify gene-level associations of rare variants that are associated with type 2 diabetes.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage–dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

Monkol Lek, Konrad J Karczewski, Eric V Minikel, Kaitlin E Samocha, Eric Banks, Timothy Fennell, Anne H O'Donnell-Luria, James S Ware, Andrew J Hill, Beryl B Cummings, Taru Tukiainen, Daniel P Birnbaum, Jack A Kosmicki, Laramie E Duncan, Karol Estrada, Fengmei Zhao, James Zou, Emma Pierce-Hoffman, Joanne Berghout, David N Cooper, Nicole Deflaux, Mark DePristo, Ron Do, Jason Flannick, Menachem Fromer, Laura Gauthier, Jackie Goldstein, Namrata Gupta, Daniel Howrigan, Adam Kiezun, Mitja I Kurki, Ami Levy Moonshine, Pradeep Natarajan, Lorena Orozco, Gina M Peloso, Ryan Poplin, Manuel A Rivas, Valentin Ruano-Rubio, Samuel A Rose, Douglas M Ruderfer, Khalid Shakir, Peter D Stenson, Christine Stevens, Brett P Thomas, Grace Tiao, Maria T Tusie-Luna, Ben Weisburd, HongHee Won, Dongmei Yu, David M Altshuler, Diego Ardissino, Michael Boehnke, John Danesh, Stacey Donnelly, Roberto Elosua, Jose C Florez, Stacey B Gabriel, Gad Getz, Stephen J Glatt, Christina M Hultman, Sekar Kathiresan, Markku Laakso, Steven McCarroll, Mark I McCarthy, Dermot McGovern, Ruth McPherson, Benjamin M Neale, Aarno Palotie, Shaun M Purcell, Danish Saleheen, Jeremiah M Scharf, Pamela Sklar, Patrick F Sullivan, Jaakko Tuomilehto, Ming T Tsuang, Hugh C Watkins, James G Wilson, Mark J Daly, Daniel G MacArthur, Hanna E Abboud, Goncalo Abecasis, Carlos A Aguilar-Salinas, Olimpia Arellano-Campos, Gil Atzmon, Ingvild Aukrust, Cathy L Barr, Graeme I Bell, Graeme I Bell, Sarah Bergen, Lise Bjørkhaug, John Blangero, Donald W Bowden, Cathy L Budman, Noël P Burtt, Federico Centeno-Cruz, John C Chambers, Kimberly Chambert, Robert Clarke, Rory Collins, Giovanni Coppola, Emilio J Córdova, Maria L Cortes, Nancy J Cox, Ravindranath Duggirala, Martin Farrall, Juan C Fernandez-Lopez, Pierre Fontanillas, Timothy M Frayling, Nelson B Freimer, Christian Fuchsberger, Humberto García-Ortiz, Anuj Goel, María J Gómez-Vázquez, María E González-Villalpando, Clicerio González-Villalpando, Marco A Grados, Leif Groop, Christopher A Haiman, Craig L Hanis, Craig L Hanis, Andrew T Hattersley, Brian E Henderson, Jemma C Hopewell, Alicia Huerta-Chagoya, Sergio Islas-Andrade, Suzanne BR Jacobs, Shapour Jalilzadeh, Christopher P Jenkinson, Jennifer Moran, Silvia JiménezMorale, Anna Kähler, Robert A King, George Kirov, Jaspal S Kooner, SUPPLEMENTARY INFORMATION doi:10.1038/nature19057

IMPORTANCE Latino populations have one of the highest prevalences of type 2 diabetes worldwide.OBJECTIVES To investigate the association between rare protein-coding genetic variants and prevalence of type 2 diabetes in a large Latino population and to explore potential molecular and physiological mechanisms for the observed relationships.DESIGN, SETTING, AND PARTICIPANTS Whole-exome sequencing was performed on DNA samples from 3756 Mexican and US Latino individuals (1794 with type 2 diabetes and 1962 without diabetes) recruited from 1993 to 2013. One variant was further tested for allele frequency and association with type 2 diabetes in large multiethnic data sets of 14 276 participants and characterized in experimental assays.MAIN OUTCOME AND MEASURES Prevalence of type 2 diabetes. Secondary outcomes included age of onset, body mass index, and effect on protein function.RESULTS A single rare missense variant (c.1522G>A [p.E508K]) was associated with type 2 diabetes prevalence (odds ratio [OR], 5.48; 95% Cl, 2.83-10.61; P = 4.4 x 10(-7)) in hepatocyte nuclear factor 1-a (HNF1A), the gene responsible for maturity onset diabetes of the young type 3 (MODY3). This variant was observed in 0.36% of participants without type 2 diabetes and 2.1% of participants with it. In multiethnic replication data sets, the p.E508K variant was seen only in Latino patients (n = 1443 with type 2 diabetes and 1673 without it) and was associated with type 2 diabetes (OR, 4.16; 95% Cl, 1.75-9.92; P = .0013). In experimental assays, HNF-1A protein encoding the p.E508K mutant demonstrated reduced transactivation activity of its target promoter compared with a wild-type protein. In our data, carriers and noncarriers of the p.E508K mutation with type 2 diabetes had no significant differences in compared clinical characteristics, including age at onset. The mean (SD) age for carriers was 45.3 years (11.2) vs 47.5 years (11.5) for noncarriers (P = .49) and the mean (SD) BMI for carriers was 28.2 (5.5) vs 29.3 (5.3) for noncarriers (P = .19).CONCLUSIONS AND RELEVANCE Using whole-exome sequencing, we identified a single low-frequency variant in the MODY3-causing gene HNF1A that is associated with type 2 diabetes in Latino populations and may affect protein function. This finding may have implications for screening and therapeutic modification in this population, but additional studies are required.

As the sample size in cancer genome studies increases, the list of genes identified as significantly mutated is likely to include more false positives; here, this problem is identified as stemming largely from mutation heterogeneity, and a new analytical methodology designed to overcome this problem is described. Cancer genomic approaches have identified scores of genes responsible for the initiation and progression of cancer. But as the sample sizes increase, the list of putatively significant genes identified by current analytical methods continues to grow and is likely to include many false positives. This study shows that this situation stems largely from mutational heterogeneity and presents a novel methodology, MutSigCV, that overcomes the problem by incorporating mutational heterogeneity into the analysis. Application of MutSigCV to more than 3,000 tumour samples from 27 different tumour types shows that mutation frequencies vary more than 1,000-fold between extreme samples both between and within tumour types. And when applied to a data set on lung cancer, MutSigCV reduced the list of significantly mutated genes from 450 to a more manageable 11, most of them previously reported to be mutated in squamous cell lung cancer. Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer1,2,3,4,5,6,7,8,9. These studies involve the sequencing of matched tumour–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour–normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.

Cervical cancer is responsible for 10-15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma-normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour-normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

Abstract Background: Cervical cancer is a major public health problem worldwide. The etiological role of human papilloma virus (HPV) infections in cervical cancer is well established. However, HPV infection is insufficient to account for the development of cervical cancers because only 2 % of women infected with HPV eventually develop invasive carcinomas. We have therefore initiated a large scale sequencing effort to provide comprehensive data on the global landscape of genomic aberrations and HPV variants that contribute to cervical cancer. Methods: We have carried out comprehensive whole exome sequencing analyses on 120 tumor-normal paired samples from Mexico and Norway. We also carried out transcriptome and whole genome sequencing analyses on a subset of the patients (81 and 14 individuals respectively). Results: The aggregate mutation rate across the dataset was 3.8 per megabase (Mb), with the most common mutations being C to T/G in the Tp*C dinucleotide context, at a rate of 15 mutations per Mb. In all, 18,037 mutations were found across the entire dataset, including 11,536 missense, 984 nonsense, 4691 silent, 231 splice site, 32 translation start site mutations, as well 443 deletions and 142 insertions. MutSig analyses to identify genes that were mutated at statistically significant frequencies across our dataset revealed 11 genes to be recurrently mutated with a false discovery rate of q<0.1 after correction for multiple hypothesis testing (and RNASeq-based evidence of robust gene expression). The most significantly mutated genes encode for members of the PIK3CA/PTEN and RAS/RAF/MAPK signaling pathways, as well as the major histocompatibility complex (MHC). We have also uncovered novel patterns of HPV transcript abundance and sites of recurrent HPV integration in cell cycle related genes. In addition, our whole genome sequencing data suggests that HPV-negative p53-mutant tumors harbor high frequencies of genomic rearrangements. Conclusion: The comprehensive catalogue of genomic alterations provided by this project reveals potential novel therapeutic targets in cervical carcinomas. Our data also sets the stage for improving diagnostic and preventive strategies, especially in resource-limited settings with the highest incidence of cervical cancer. Citation Format: Akinyemi I. Ojesina, Lee Lichtenstein, Lauren Ambrogio, Kristian Cibulskis, Samuel Freeman, Chandra Sekhar Pedamallu, Bjørn Bertelsen, Ivan Imaz, Karla Vazquez, Alberto Salido Guadarrama, Victor Treviño, Sandra Romero-Cordoba, Fujiko Duke, Bethany Kaplan, Iram Rodriguez, Magali Espinosa Castilla, Katherine Woie, Line Bjorge, Elisabeth Wik, Mari K. Halle, Erling Høivik, Camilla Krakstad, Gabriela Gómez Macías, María de Lourdes Garza Rodríguez, Jorge Vazquez, Carlos Rodea, Adrian Cravioto, Maria L. Cortes, Heidi Greulich, Christopher P. Crum, Lars Akslen, Hugo Barrera Saldaña, Jorge Melendez-Zajgla, Gad Getz, Helga B. Salvesen, Matthew L. Meyerson. Landscape of human and viral genomic alterations in cervical carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4604. doi:10.1158/1538-7445.AM2013-4604

Abstract Today, more than 55% of the world's breast cancer cases are diagnosed in low and middle-income countries and in 2020, more that 70% of the cases will come from the developing nations. In Mexico, breast cancer-specific mortality doubled during the past 20 years, representing the second-leading cause of death in women between 30 and 59 years and the leading cause of cancer related death in the female population. According to statistics, in Mexico a woman dies due to breast cancer every two hours. Even though breast cancer represents a major public health problem in the developing world, knowledge about the genetic and genomic structure of breast tumors in Mexican or Latin American populations is very limited. In the past four years, we have participated in the Slim Initiative of Genomic Medicine (SIGMA) Project, a collaboration between the Carlos Slim Institute of Health, the Broad Institute, and the National Institute of Genomic Medicine in Mexico city. The goal of the SIGMA project is to characterize the genomic basis of common diseases, including several types of cancer. This effort has focused on the application of whole genome and whole exome sequencing of human tumors. In the case of breast cancer, we have analyzed the whole genomes of 22 tumor/normal tissue pairs and the whole exomes of 103 tumor/normal tissues from Mexican and Vietnamese patients. Sequence analysis led to the novel identification of potential loss of function mutations of the CBFB transcription factor, and deletions of its partner RUNX1, an event which has never been previously reported in breast tumors or in any other epithelial tumor. Of clinical relevance, we also identified a somatic translocation involving MAGI3 and AKT3 in a triple negative breast tumor. Ectopic expression of the fusion transcrip leads to constitutive phosphorylation of downstream GSK and loss of contact inhibition. Most importantly, the activity of the fusion protein can be abrogated by an ATP-competitive small molecule inhibitor of AKT, potentially representing a new therapeutic avenue for these patients. In parallel with sequencing, we have also been working on the analysis of somatic DNA copy number aberrations, messenger RNA expression, and microRNA expression patterns in tumors from Mexican patients. Intrinsic breast cancer sub-typing in 125 tumors from Mexican patients showed that 13.6% of the tumors were basal-like, 16.8% were Her2-enriched, 24.8% Luminal A, 34.4% Luminal B and 10.4 normal-like. With microRNA expression, we have identified a group of microRNAs whose role in breast cancer has not been previously described and are currently analyzing differential microRNA expression across tumor sub-types, in particular triple negative tumors, where we have been able to identify at least three different tumor sub-groups based on microRNA expression patterns. Citation Format: Shantanu Banerji, Kristian Cibulskis, Claudia Rangel-Escareño, Kristin K. Brown, Scott L. Carter, Abbie M. Frederick, Michael S. Lawrence, Andrey Y. Sivachenko, Carrie Sougnez, Lihua Zou, Maria L. Cortes, Juan C. Fernandez-Lopez, Shouyong Peng, Kristin G. Ardlie, Daniel Auclair, Veronica Bautista-Piña, Fujiko Duke, Joshua Francis, Joonil Jung, Antonio Maffuz-Aziz, Robert C. Onofrio, Melissa Parkin, Nam H. Pho, Valeria Quintanar-Jurado, Alex H. Ramos, Rosa Rebollar-Vega, Sergio A. Rodríguez-Cuevas, Sandra L. Romero-Cordoba, Steven E. Schumacher, Nicolas Stransky, Kristin M. Thompson, Laura Uribe-Figueroa, Jose Baselga, Rameen Beroukhim, Kornelia Polyak, Dennis C. Sgroi, Andrea L. Richardson, Gerardo Jimenez-Sánchez, Eric S. Lander, Stacey B. Gabriel, Levi A. Garraway, Todd R. Golub, Jorge Meléndez-Zajgla, Alex Toker, Gad Getz, Matthew Meyerson, Alfredo Hidalgo-Miranda. Molecular profiling of breast cancer in Mexico: Identification of novel therapeutic targets through whole genome sequencing analysis. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL07-01.

This paper reports one of the largest breast cancer whole-exome and whole-genome sequencing efforts so far, identifying previously unknown recurrent mutations in CBFB, deletions of RUNX1 and recurrent MAGI1–AKT3 fusion; the fusion suggests that the use of ATP-competitive AKT inhibitors should be evaluated in clinical trials. This paper reports one of the largest whole-exome sequencing efforts in human breast cancers so far, complemented by whole-genome sequences of 22 breast cancer/normal pairs. The authors analysed diverse subtypes from patients in Mexico and Vietnam and identified recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1, as well as a recurrent MAGI3–AKT3 fusion enriched in triple-negative breast cancers (those lacking oestrogen and progesterone receptors and ERBB2 expression). The fusion leads to constitutive activation of AKT kinase, which can be counteracted by treatment with a small-molecule inhibitor. Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone1. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy2,3,4. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration5. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements6,7,8,9,10. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA11, TP536, AKT112, GATA313 and MAP3K110, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3–AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3–AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Head and neck squamous cell carcinoma (HNSCC) is a common, morbid, and frequently lethal malignancy. To uncover its mutational spectrum, we analyzed whole-exome sequencing data from 74 tumor-normal pairs. The majority exhibited a mutational profile consistent with tobacco exposure; human papillomavirus was detectable by sequencing DNA from infected tumors. In addition to identifying previously known HNSCC genes (TP53, CDKN2A, PTEN, PIK3CA, and HRAS), our analysis revealed many genes not previously implicated in this malignancy. At least 30% of cases harbored mutations in genes that regulate squamous differentiation (for example, NOTCH1, IRF6, and TP63), implicating its dysregulation as a major driver of HNSCC carcinogenesis. More generally, the results indicate the ability of large-scale sequencing to reveal fundamental tumorigenic mechanisms.

Abstract Head and neck cancer is responsible for an enormous cancer burden worldwide, with an estimated 500,000 new cases per year. However, knowledge of the spectrum of genetic alterations that drive this malignancy remains incomplete. Previous studies of this disease have identified recurrent somatic mutations in a few specific genes, but few systematic genome-scale studies have been performed. Accordingly, we have undertaken a pilot study of complete genome/exome characterization in this malignancy. We obtained genomic DNA from 40 head and neck tumors together with matched normal DNA from whole blood. To date, we have obtained whole exome sequencing of 28 tumor/normal pairs and whole genome sequencing of two tumor/normal pairs. The average base mutation rate is 3.1 × 10-6 (range = 2.9 × 10-7 to 8.8 × 10-6), consistent with rates observed in lung cancer and other tobacco-related malignancies. We also identified 24 high-confidence somatic rearrangements in one sample and 120 in the other sample (including 7 and 44 inter-chromosomal rearrangements, respectively). In addition to frequent mutations of TP53 (53%), HRAS, PIK3CA, and PTEN, we also observed other recurrently mutated genes that implicate several oncogenic pathways. Furthermore, layering of somatic mutation data onto known pathway information strengthens the evidence for several oncogenic processes not previously implicated in head and neck cancer. Altogether, these data should enhance our understanding of the mechanisms that drive head and neck carcinogenesis and progression and may also illuminate new therapeutic opportunities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2011-3931

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and sensitivity to ionizing radiation (IR). We have previously shown that a herpes simplex virus type 1 (HSV-1) amplicon vector carrying the human ataxia-telangiectasia mutated (ATM) complementary DNA (cDNA) is able to correct aspects of the cellular phenotype of human A-T cells in culture, and is also able to transfer the ATM cDNA to the Atm(-/-) mouse cerebellum. In order to achieve stable gene replacement, we have generated an HSV/adeno-associated virus (AAV) hybrid amplicon vector carrying the expression cassettes for the ATM cDNA [(9.2 kilobases (kb)] and enhanced green fluorescent protein (EGFP), flanked by AAV inverted terminal repeats (ITRs). This hybrid vector, in the presence of AAV Rep proteins, mediates site-specific integration into the AAVS1 site on chromosome 19 in human cells and in Atm(-/-) mice carrying that human locus. The functional activity of the vector-derived ATM was confirmed in vitro and in vivo by ATM autophosphorylation at Ser-1981 after IR. This proof-of-principle study establishes the ability of HSV/AAV hybrid amplicon vectors to mediate functional targeted integration of the ATM cDNA into A-T cells in culture and in Atm(-/-) mice in vivo, thus laying a foundation for possible gene therapy approaches in the treatment of A-T patients.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. The large size of the cDNA (9.2 kb) required to correct the hereditary deficiency in A-T has limited use of many vector types for genetic treatment. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. We have previously reported the construction of an HSV-1 amplicon vector carrying the full-length human ATM cDNA, and shown the ability of this vector to correct aspects of the cellular phenotype of human A-T cells in culture. In order to restore stable gene function in A-T cells, it would be optimal to employ vectors that have the capacity for efficient genomic integration in a site-specific manner. We have generated an HSV/ AAV hybrid amplicon vector carrying the ATM cDNA flanked with AAV ITR sequences, which should be able, in the presence of AAV Rep proteins, to mediate site-specific recombination into the AAVS1 site on chromosome 19 in human cells. The hybrid vector, designated as pHyRIGN-ATM, contains the ATM cDNA under the CBA promoter, the GFP reporter gene under the CMV promoter, and a neomycin resistance cassette, all between the ITRs. The integrity of the amplicon construct has been verified by restriction analysis and sequencing. To characterize integration sites in transduced clones we have used human 293 control cells, and SV40-transformed human fibroblast lines from a healthy A-T heterozygote and a patient with A-T. Following infection with the pHyRIGN-ATM hybrid amplicon vector, or a hybrid control vector, stable clones expressing EGFP have been selected with G418. Positive clones have been expanded and evaluated for FLAG- ATM expression. Using immunoprecipitation with FLAG M2 antibody and western blot analysis with ATM antibody, we have been able to detect FLAG-tagged ATM protein in the A-T clones transduced with the pHyRIGN-ATM hybrid amplicon vector. In addition, we determined the functional activity of the vector- derived FLAG-ATM by autophosphorylation of ATM on Ser-1981 after ionizing radiation. These experiments show that clones obtained after infection with the HSV/AAV vector express functional ATM. Clones are currently being analyzed for the frequency and site of genomic integration by PCR and Southern blot analysis using different primers and probes to distinguish random versus AAVS1 integrations, and for the integration of ITR-flanked and/or amplicon backbone sequences. Transgenic mice bearing the human AAVS1 site, which has been shown to function as a target for this integration event, have been crossed to Atm +/- mice to create Atm -/- bearing the AAVS1 site. Current efforts are focused on the delivery of this hybrid vector to different tissues of Atm -/- AAVS1 +/+ mice. These mice will allow us to evaluate whether we can achieve targeted integration of the ATM cDNA into the Atm -/- genome for gene replacement in cells in vivo, thereby demonstrating the feasibility of HSV/AAV amplicon vectors as a gene therapy vector for A-T. This work was supported by A-T Children's Project and NINDS R21.

Ataxia–telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Herpes simplex virus type 1 (HSV-1) amplicon vectors provide a means to deliver large genes to the nervous system efficiently and safely. We have generated an amplicon vector, carrying human FLAG-tagged A-T mutated (ATM), as well as an enhanced green fluorescent protein (EGFP) marker gene. Due to the lack of effective and reliable antibodies for ATM and FLAG appropriate for immunohistochemistry in mouse tissue sections, expression of the human FLAG-tagged ATM was confirmed in the mouse cerebellum at the RNA level by reverse transcription followed by quantitative PCR, and by radioactive in situ hybridization. In addition, we were able to immunoprecipitate the full-length human ATM protein from the cerebella of Atm −/− mice post-infection. This vector has been injected into the cerebella of Atm −/− mice with gene delivery to thousands of cells, including Purkinje cells, based on the EGFP marker gene. The expression of human FLAG-tagged ATM has been demonstrated in the cerebella of Atm−/− mice at the transcription and translational level three days post-infection. To our knowledge, this is the first report of vector-mediated delivery of the human ATM cDNA to an Atm −/− mouse. These vectors provide the groundwork to develop gene therapy approaches for A-T patients.

The realization of gene therapy for the central nervous system has advanced to the realmsof clinical practice. The brain and spinal cord, once inaccessible to certain therapies, havebeen unlocked by gene therapy vectors allowing medical practitioners to target areas of thebrain in a manner never before attainable. For example, certain vectors can target tumors,replicate in those cells, and deliver toxic substances to rid the brain of cancers. Also, wecan now deliver genes to certain cell types of the brain and allow continuous gene delivery,delivering a therapeutic gene to the exact cells that are perishing in neurological disordersor injury. These discoveries have advanced the concept of gene therapy for the nervoussystem to a clinical reality.

Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder characterized by abnormalities in cellular migration, proliferation, and differentiation in many tissues. Benign hamartomas develop in multiple organs, believed to be caused by somatic mutation in addition to germ line mutation to cause loss of both alleles of either the TSC1 or TSC2 tumor suppressor gene, with resultant dysregulated growth due to loss of hamartin or tuberin function, respectively. This study focuses on detecting spontaneous lesions in a knockout mouse model of TSC2 by magnetic resonance imaging (MRI) and exploring the efficiency of introducing gene products into lesions, using transduced endothelial cells as gene vehicles. MRI was shown to be effective in detecting spontaneous lesions in multiple tissues as a means of assessing the prevalence of tumors. Tsc(2+/) heterozygous mice were screened at 12-24 months of age. MRI detected 100% of the renal lesions (cystadenomas, renal cell carcinomas) and 75% of the hepatic lesions (hemangiosarcomas), later identified by histology. Cell-mediated gene delivery was evaluated by immunohistochemical analysis of renal, hepatic, and lung lesions after intravenous delivery of MS1 mouse endothelial cells, transduced to express an enhanced form of green fluorescent protein (EGFP). Preliminary immunohistochemical analysis, using a polyclonal antibody to EGFP and a horseradish peroxidase-diaminobenzidine detection system, revealed these cells throughout liver, kidney, and lung sections from injected animals, organs that are frequently affected in TSC2 patients, as well as within the lesions themselves.

It has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood.

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.