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1.  Common Genetic Variation in the 3-BCL11B Gene Desert Is Associated With Carotid-Femoral Pulse Wave Velocity and Excess Cardiovascular Disease Risk The AortaGen Consortium 
Mitchell, Gary F. | Verwoert, Germaine C. | Tarasov, Kirill V. | Isaacs, Aaron | Smith, Albert V. | Yasmin | Rietzschel, Ernst R. | Tanaka, Toshiko | Liu, Yongmei | Parsa, Afshin | Najjar, Samer S. | O’Shaughnessy, Kevin M. | Sigurdsson, Sigurdur | De Buyzere, Marc L. | Larson, Martin G. | Sie, Mark P.S. | Andrews, Jeanette S. | Post, Wendy S. | Mattace-Raso, Francesco U.S. | McEniery, Carmel M. | Eiriksdottir, Gudny | Segers, Patrick | Vasan, Ramachandran S. | van Rijn, Marie Josee E. | Howard, Timothy D. | McArdle, Patrick F. | Dehghan, Abbas | Jewell, Elizabeth | Newhouse, Stephen J. | Bekaert, Sofie | Hamburg, Naomi M. | Newman, Anne B. | Hofman, Albert | Scuteri, Angelo | De Bacquer, Dirk | Ikram, Mohammad Arfan | Psaty, Bruce | Fuchsberger, Christian | Olden, Matthias | Wain, Louise V. | Elliott, Paul | Smith, Nicholas L. | Felix, Janine F. | Erdmann, Jeanette | Vita, Joseph A. | Sutton-Tyrrell, Kim | Sijbrands, Eric J.G. | Sanna, Serena | Launer, Lenore J. | De Meyer, Tim | Johnson, Andrew D. | Schut, Anna F.C. | Herrington, David M. | Rivadeneira, Fernando | Uda, Manuela | Wilkinson, Ian B. | Aspelund, Thor | Gillebert, Thierry C. | Van Bortel, Luc | Benjamin, Emelia J. | Oostra, Ben A. | Ding, Jingzhong | Gibson, Quince | Uitterlinden, André G. | Abecasis, Gonçalo R. | Cockcroft, John R. | Gudnason, Vilmundur | De Backer, Guy G. | Ferrucci, Luigi | Harris, Tamara B. | Shuldiner, Alan R. | van Duijn, Cornelia M. | Levy, Daniel | Lakatta, Edward G. | Witteman, Jacqueline C.M.
Background
Carotid-femoral pulse wave velocity (CFPWV) is a heritable measure of aortic stiffness that is strongly associated with increased risk for major cardiovascular disease events.
Methods and Results
We conducted a meta-analysis of genome-wide association data in 9 community-based European ancestry cohorts consisting of 20,634 participants. Results were replicated in 2 additional European ancestry cohorts involving 5,306 participants. Based on a preliminary analysis of 6 cohorts, we identified a locus on chromosome 14 in the 3′-BCL11B gene desert that is associated with CFPWV (rs7152623, minor allele frequency = 0.42, beta=−0.075±0.012 SD/allele, P = 2.8 x 10−10; replication beta=−0.086±0.020 SD/allele, P = 1.4 x 10−6). Combined results for rs7152623 from 11 cohorts gave beta=−0.076±0.010 SD/allele, P=3.1x10−15. The association persisted when adjusted for mean arterial pressure (beta=−0.060±0.009 SD/allele, P = 1.0 x 10−11). Results were consistent in younger (<55 years, 6 cohorts, N=13,914, beta=−0.081±0.014 SD/allele, P = 2.3 x 10−9) and older (9 cohorts, N=12,026, beta=−0.061±0.014 SD/allele, P=9.4x10−6) participants. In separate meta-analyses, the locus was associated with increased risk for coronary artery disease (hazard ratio [HR]=1.05, confidence interval [CI]=1.02 to 1.08, P=0.0013) and heart failure (HR=1.10, CI=1.03 to 1.16, P=0.004).
Conclusions
Common genetic variation in a locus in the BCL11B gene desert that is thought to harbor one or more gene enhancers is associated with higher CFPWV and increased risk for cardiovascular disease. Elucidation of the role this novel locus plays in aortic stiffness may facilitate development of therapeutic interventions that limit aortic stiffening and related cardiovascular disease events.
doi:10.1161/CIRCGENETICS.111.959817
PMCID: PMC3288392  PMID: 22068335
aorta; arterial stiffness; pulse wave velocity; genetics; cardiovascular disease
2.  Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform 
BMC Medical Genomics  2012;5:17.
Background
Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.
Results
In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.
Conclusions
We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.
doi:10.1186/1755-8794-5-17
PMCID: PMC3443074  PMID: 22607986
Deafness; Next generation sequencing; PCR based enrichment; Genetic diagnostics
3.  Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics 
PLoS ONE  2011;6(9):e25531.
Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics.
doi:10.1371/journal.pone.0025531
PMCID: PMC3184136  PMID: 21980484
4.  Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline 
BMC Bioinformatics  2010;11:269.
Background
Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions.
Results
We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data.
Conclusions
We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.
doi:10.1186/1471-2105-11-269
PMCID: PMC2880033  PMID: 20487544
5.  PubMeth: a cancer methylation database combining text-mining and expert annotation 
Nucleic Acids Research  2007;36(Database issue):D842-D846.
Epigenetics, and more specifically DNA methylation is a fast evolving research area. In almost every cancer type, each month new publications confirm the differentiated regulation of specific genes due to methylation and mention the discovery of novel methylation markers. Therefore, it would be extremely useful to have an annotated, reviewed, sorted and summarized overview of all available data. PubMeth is a cancer methylation database that includes genes that are reported to be methylated in various cancer types. A query can be based either on genes (to check in which cancer types the genes are reported as being methylated) or on cancer types (which genes are reported to be methylated in the cancer (sub) types of interest). The database is freely accessible at http://www.pubmeth.org.
PubMeth is based on text-mining of Medline/PubMed abstracts, combined with manual reading and annotation of preselected abstracts. The text-mining approach results in increased speed and selectivity (as for instance many different aliases of a gene are searched at once), while the manual screening significantly raises the specificity and quality of the database. The summarized overview of the results is very useful in case more genes or cancer types are searched at the same time.
doi:10.1093/nar/gkm788
PMCID: PMC2238841  PMID: 17932060

Results 1-5 (5)