Showing papers on "Exome sequencing published in 2007"

Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing.

Abstract: We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (<20) typically analyzed by traditional bisulfite sequencing. Comprehensive analysis of CpG methylation patterns at a single DNA molecule level using clustering algorithms revealed differential methylation patterns between diseases. A significant increase in methylation was detected in ALL and FL samples compared with CLL and MCL. Furthermore, a progressive spreading of methylation was detected from the periphery toward the center of select CpG islands in the ALL and FL samples. The ultradeep sequencing also allowed simultaneous analysis of genetic and epigenetic data and revealed an association between a single nucleotide polymorphism and the methylation present in the LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes.

Selected Exonic Sequencing of the AGXT Gene Provides a Genetic Diagnosis in 50% of Patients with Primary Hyperoxaluria Type 1

Abstract: Background: Definitive diagnosis of primary hyperoxaluria type 1 (PH1) requires analysis of alanine:glyoxylate aminotransferase (AGT) activity in the liver. We have previously shown that targeted screening for the 3 most common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) can provide a molecular diagnosis in 34.5% of PH1 patients, eliminating the need for a liver biopsy. Having reviewed the distribution of all AGXT mutations, we have evaluated a diagnostic strategy that uses selected exon sequencing for the molecular diagnosis of PH1. Methods: We sequenced exons 1, 4, and 7 for 300 biopsy-confirmed PH1 patients and expressed the identified missense mutations in vitro. Results: Our identification of at least 1 mutation in 224 patients (75%) and 2 mutations in 149 patients increased the diagnostic sensitivity to 50%. We detected 29 kinds of sequence changes, 15 of which were novel. Four of these mutations were in exon 1 (c.2\_3delinsAT, c.30\_32delCC, c.122G>A, c.126delG), 7 were in exon 4 (c.447_454delGCTGCTGT, c.449T>C, c.473C>T, c.481G>A, c.481G>T, c.497T>C, c.424-2A>G), and 4 were in exon 7 (c.725insT, c.737G>A, c.757T>C, c.776 + 1G>A). The missense changes were associated with severely decreased AGT catalytic activity and negative immunoreactivity when expressed in vitro. Missense mutation c.26C>A, previously described as a pathological mutation, had activity similar to that of the wild-type enzyme. Conclusions: Selective exon sequencing can allow a definitive diagnosis in 50% of PH1 patients. The test offers a rapid turnaround time (15 days) with minimal risk to the patient. Demonstration of the expression of missense changes is essential to demonstrate pathogenicity.

Analysis of large-scale sequencing of small RNAs.

Abstract: The advent of large-scale sequencing has opened up new areas of research, such as the study of Piwi-interacting small RNAs (piRNAs). piRNAs are longer than miRNAs, close to 30 nucleotides in length, involved in various functions, such as the suppression of transposons in germline. Since a large number of them (many tens of thousands) are generated from a wide range of positions in the genome, large-scale sequencing is the only way to study them. The key to understanding their genesis and biological roles is efficient analysis, which is complicated by the large volumes of sequence data. Taking account of the underlying biology is also important. We describe here novel analyses techniques and tools applied to small RNAs from germ cells in D. melanogaster, that allowed us to infer mechanism and biological function.

The first large duplication of the RSK2 gene identified in a Coffin-Lowry syndrome patient.

Abstract: Heterogeneous mutations in the X-linked gene RPS6KA3, encoding the protein kinase RSK2, are responsible for Coffin-Lowry Syndrome. Here we have further studied a male patient with a highly suggestive clinical diagnosis of CLS but in whom no mutation was found by exon sequencing. Western blot analysis revealed a protein much larger than the normal expected size. Sequencing of the RSK2 cDNA, showed the presence of an in-frame tandem duplication of exons 17–20. The mutated RSK2 protein was found to be inactive in an in-vitro kinase assay. This event, which was the result of a homologous unequal recombination between Alu sequences, is the first reported large duplication of the RPS6KA3 gene. Our finding provides further evidence that immunoblot analysis, or a molecular assay capable to detect large genomic mutational events, is essential for patients with a highly suggestive CLS clinical diagnosis but remaining without mutation after exon sequencing.

HLA-DQB1 sequencing-based typing updated.

Abstract: The use of direct sequencing as a typing strategy is well acknowledged. Direct sequencing identifies all sequence motifs including new polymorphisms in heterozygous sequences. The earlier protocols for human leukocyte antigen HLA-DQB1 Sequencing-Based Typing (SBT) frequently encounter preferential amplification of one of the alleles that can lead to unreliable sequences or even to allelic dropout. In our new approach, the quality of the exon 2 sequences, now including both alleles to the same extend, was achieved by amplifying the HLA-DQB1*05/06 group into two groups by changing the common 3' amplification primer. In combination with exon 3 this updated HLA-DQB1 protocol provides a reliable approach for heterozygous sequencing.

Statistical methods for the analysis of cancer genome sequencing data

Abstract: The purpose of cancer genome sequencing studies is to determine the nature and types of alterations present in a typical cancer and to discover genes mutated at high frequencies. In this article we discuss statistical methods for the analysis of data generated in these studies. We place special emphasis on a two-stage study design introduced by Sjoblom et al.[1]. In this context, we describe statistical methods for constructing scores that can be used to prioritize candidate genes for further investigation and to assess the statistical significance of the candidates thus identified. 1 Hosted by The Berkeley Electronic Press

Tomato genome sequencing: deciphering the euchromatin region of the chromosome 8

Abstract: As a member of the Solanaceae Genomics Network International Tomato Sequencing Project, we launched on sequencing of the chromosome 8. Our task is to sequence the euchromatin region of the chromosome, the estimated size of which is 17 megabases. BAC-by-BAC strategy is adopted for sequencing. We initially received BAC clone candidates anchored to overgo probes developed from 33 markers mapped on chromosome 8. For confirmation of the BAC candidates, we analyzed the sequence of PCR product amplified from the BAC DNA with primers designed on the marker sequence. Twenty-five BAC clones were verified and subjected to shotgun sequencing. As of Nov. 2006, we finished 40 clones to Phase 3 (total non-redundant length 4,429,168 bases) of 25 are seed and 15 are extended clones. In order to find additional seed points, we performed PCR screening of markers against 3D DNA-pool of LE-HBa, SL-MboI and SL-EcoRI BAC libraries, and succeeded in obtaining 9 new seeds. We report the current status of our project and future perspectives toward the final goal, which include development of new microsatellite markers from tomato EST sequences and Selected BAC Mixture shotgun sequencing for the gap filling.

Pyrosequencing Technology for Short DNA Sequencing and Whole Genome Sequencing

Abstract: Recent impressive advances in DNA sequencing technologies have accelerated the detailed analysis of genomes from many organisms. We have been observing reports of complete or draft versions of the genome sequence of several well-studied, multicellular organisms. Human biology and medicine are in the midst of a revolution by Human Genome Project1) as the main catalyst. The chain termination sequencing method, also known as Sanger sequencing, developed by Frederick Sanger and colleagues2), has been the most widely used sequencing method since its advent in 1977 and still is in use after more than 29 years since its advent. The remarkable advances in chemistry and automation to the Sanger sequencing method has made it to a simple and elegant technique, central to almost all past and current genome-sequencing projects of any significant scale. Despite all these grand advantages, there are limitations in this method, which could be complemented with other techniques. Among the current state-of-art DNA sequencing techniques, Pyrosequencing3) has emerged, which is being used for a wide variety of applications. In the beginning, the method was only restricted to single nucleotide polymorphisms (SNP) genotyping4), 5) and short reads6), 7) when it was introduced in 1997 but it is now being used for broader applications. 2.Pyrosequencing chemistry

Genetic Studies of Familial Myeloproliferative Disorders

Abstract: Hereditary thrombocythemia (HT) is an autosomal dominant disorder with clinical features resembling sporadic essential thrombocythemia. HT families share similar clinical symptoms caused by heterogeneous genetic alterations. Inherited germ-line mutations in the thrombopoietin (TPO) gene and its receptor MPL have been found causing thrombocytosis in a number of HT families. Five reported mutations in the thrombopoietin gene are all located in the 5 prime untranslated region (5’UTR) and cause overproduction of Tpo protein by the same mechanism: increased translation efficiency for the mutant mRNAs. One mutation identified in the MPL gene is located at the transmembrane domain and results in a hyperactive receptor, thereby leading to thrombocytosis. All these germ-line mutations have not been found in sporadic patients and are only responsible for the etiology of some HT families, indicating that the occurrence of these germ-line mutations is a rare event. The disease-causing genes for many HT families remain unknown. Identifying genetic lesions in these families will increase our knowledge of the physiology of thrombopoiesis and some of these unknown genetic components may contribute to the pathogenesis in sporadic MPD patients. In the first part of the project for genetic studies of HT families, the TPO and MPL genes were analyzed by genomic DNA exon sequencing and linkage analysis. A splice donor mutation in the TPO gene was identified in a Polish family. This mutation was previously identified in a Dutch family and the reoccurrence of this rare mutation has not been reported to date. In order to determine whether the 6 mutation mutation in these two families arose de novo or from a founder effect, haplotype analysis was performed to examine polymorphic DNA sequences in the vicinity of the mutation using microsatellites and single nucleotide polymorphism (SNP) in these two families. Six microsatellite markers on the affected allele showed different sizes in PCR products and 3 SNPs close to the mutation differed in their sequences between the two families. We therefore concluded that the mutation in these two families occurred de novo. The previously reported MPL mutation at the transmembrane domain of MPL protein was identified in one of the HT families studied here. Recently, 5 additional HT families were found carrying this mutation. We conducted haplotype analysis using microsatellite markers in the MPL gene locus for the 6 HT families. Four microsatellite markers surrounding the MPL mutation showed identical sizes in the PCR products on the affected allele, suggesting that the MPL mutation occurred from a single founder event. This may explain the high frequency of this mutation in HT families. In a large US family with HT, where the TPO and MPL genes were excluded as disease causes, genome-wide linkage analysis was performed aiming to identify novel genetic component for the thrombocytosis phenotype. Two genetic regions with significant logarithm of odds (LOD) score values have been located using microsatellites and SNP chip arrays. Candidate gene sequencing revealed one novel polymorphism in the gelsolin gene, which encodes an actin-binding protein abundant in platelets. Gelsolin has multiple biological functions in addition to cytoskeletal actin modulation. Functional studies in cell proliferation assays and mouse bone marrow transplantation did not validate this polymorphism as an active disease causing mutation. Further studies on this polymorphism in platelet biogenesis are planned for the future. In addition, sequencing of all the candidate genes in the segregating regions is in progress. In a second project, genome-wide linkage analyses were performed using microsatellites and SNP chip arrays in a family with secondary polycythemia inherited in an autosomal recessive mode. Both parametric and nonparametric linkage analysis were conducted for this family. Five genetic regions were found linked to the disease phenotype. A few candidate genes were sequenced and studied, however no genetic variation was found so far. Additionally, no mutations were found in several genes involved in erythropoiesis and oxygen sensing pathway. Burst forming units-Erythroid cultures in hypoxia condition showed high expression of the EPO gene in 3 out of 4 affected family members, suggesting a potential unknown defect in the oxygen-sensing pathway.