There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for approximately 10(6) single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

/pdf/core-signaling-pathways-in-human-pancreatic-cancers-revealed-2l25lcwhrc.pdf

Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses

Identification and characterization of a spinal muscular atrophy-determining gene

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore

PL02-05 

All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

Patterns of Somatic Mutation in Human Cancer Genomes

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.

/pdf/a-novel-method-for-real-time-quantitative-rt-pcr-e4vb7sbeq0.pdf

A novel method for real time quantitative RT-PCR.

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.

/pdf/aggresomes-a-cellular-response-to-misfolded-proteins-3b51qi1k8m.pdf

Aggresomes: A Cellular Response to Misfolded Proteins

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.

The "megaprimer" method of site-directed mutagenesis.

Purpose A clinical testing cohort was used to gain a broader understanding of the spectrum of tumors associated with germline p53 mutations to aid clinicians in identifying high-risk families. Patients and Methods Full sequencing of the coding exons (2 to 11) and associated splice junctions of the p53 gene was performed on 525 consecutive patients whose blood samples were submitted for diagnostic testing. Clinical features of p53 germline carriers in this cohort were characterized, clinical referral schemes based on reported p53-associated family phenotypes were evaluated, and practical mutation prevalence tables were generated. Results Mutations were identified in 91 (17%) of 525 patients submitted for testing. All families with a p53 mutation had at least one family member with a sarcoma, breast, brain, or adrenocortical carcinoma (ACC). Every individual with a choroid plexus tumor (eight of eight) and 14 of 21 individuals with a childhood ACC had a mutation regardless of family history. Based on report...

https://ascopubs.org/doi/pdf/10.1200/JCO.2008.16.6959

Beyond Li Fraumeni Syndrome: Clinical Characteristics of Families With p53 Germline Mutations

MicroRNAs (miRNAs) are 21–25-nucleotide-long, noncoding RNAs that are involved in translational regulation Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants Functional assays were developed and performed on these variants In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases

/pdf/snps-in-human-mirna-genes-affect-biogenesis-and-function-1x9h3v1nyv.pdf

Steve S. Sommer

Papers

The "megaprimer" method of site-directed mutagenesis.

Beyond Li Fraumeni Syndrome: Clinical Characteristics of Families With p53 Germline Mutations

SNPs in human miRNA genes affect biogenesis and function.

Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection

Formamide can dramatically improve the specificity of PCR.