Nonlinear dimension reduction in pathway-based genome-wide association analysis

Pathway based genome wide association study evolves from pathway analysis for microarray gene expression and is under rapid development as a complementary for single-SNP based genome wide association study. However, it faces new challenges, such as the summarization of SNP statistics to pathway statistics. The current study applies the ridge regularized Kernel Sliced Inverse Regression (KSIR) to achieve dimension reduction and compared this method to the other two widely used methods, the minimal-p-value (minP) approach of assigning the best test statistics of all SNPs in each pathway as the statistics of the pathway and the principal component analysis (PCA) method of utilizing PCA to calculate the principal components of each pathway. Comparison of the three methods using simulated datasets consisting of 500 cases, 500 controls and100 SNPs demonstrated that KSIR method outperformed the other two methods in terms of causal pathway ranking and the statistical power. PCA method showed similar performance as the minP method. KSIR method also showed a better performance over the other two methods in analyzing a real dataset, the WTCCC Ulcerative Colitis dataset consisting of 1762 cases, 3773 controls as the discovery cohort and 591 cases, 1639 controls as the replication cohort. Several immune and non-immune pathways relevant to ulcerative colitis were identified by these methods. Results from the current study provided a reference for further methodology development and identified novel pathways that may be of importance to the development of ulcerative colitis.^

Genome -wide and locus -specific instability

The discovery of expanded simple repeated sequences causing or associated with human disease has lead to a new area of research involved in the elucidation of how the expanded repeat causes disease and how the repeat becomes unstable. ^ To study the genetic basis of the (CTG)n repeat instability in the DMPK gene in myotonic dystrophy (DM1) patients, somatic cell hybrids were constructed between the lymphocytes of DM1 patients and a variety of Chinese hamster ovary (CHO) cell DNA repair gene deficient mutants. By using small pool PCR (SP-PCR), the instability of the (CTG)n can be quantitated for both the frequency and sizes of length change mutations. ^ Additional SP-PCR analysis on 2/11 subclones generated from this original hybrid showed a marked increase in large repeat deletions, ∼50%. A bimodal distribution of repeats was seen around the progenitor allele and at a large deleted product (within the normal range) with no intermediate products present. ^ To determine if the repair capacity of the CHO cell led to a mutator phenotype in the hamster and hybrid clones, SP-PCR was also done on 3 hamster microsatellites in a variety of hamster cell backgrounds. No variant alleles were seen in over 2500 genome equivalents screened. ^ Human-hamster hybrids have long been shown to be chromosomally unstable, yet information about the stability of repeated sequences was not known. To test if repeat instability was associated with either intact or non-intact human chromosomes, more than 300 microsatellite repeats on 13 human chromosomes (intact and non-intact) were analyzed in eight hybrid cells. No variants were seen between the hybrid and patient alleles in the hybrids. ^ To identify whether DM1 patients have a previously undetected level of genome wide instability or if the instability is truly locus specific, SP-PCR was done on 6 human microsatellites within the patient used to make the hybrid cells. No variants were seen in over 1000 genomes screened. ^ These studies show that the somatic cell hybrid approach is a genetically stable system that allows for the determination of factors that could lead to changes in microsatellite instability. It also shows that there is something inherent about the DM1 expanded (CTG)n repeat that it is solely targeted by, as of yet, and unknown mechanism that causes the repeat to be unstable. (Abstract shortened by UMI.)^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^

Genome instability quantified in constitutive tissues as well as putatively stable tumor tissue in hereditary non -polyposis colon cancer patients by small pool PCR

Microsatellite instability (MSI) is a hallmark of the mutator phenotype associated with Hereditary Non-Polyposis Colon Cancer (HNPCC). The MSI-High (MSI-H) HNPCC population has been well characterized, but the microsatellite low and stable (MSI-L/MSS) HNPCC population is much less understood. We hypothesize there are significant levels of MSI in HNPCC DNA classified as MSI-L/MSS, but no single variant allele makes up a sufficient population in the tumor DNA to be detected by standard analysis. Finding variants would suggest there is a mutator phenotype for the MSI-L/MSS HNPCC population that is distinct from the MSI-H HNPCC populations. This study quantified and compared MSI in HNPCC patients previously shown to be MSI-H, MSI-L/MSS and an MSI-H older, sporadic colorectal cancer patient. Small-pool Polymerase Chain Reactions (SP-PCRs) were conducted where the DNAs from each sample and controls are diluted into multiple pools, each containing approximately single genome equivalents. At least 100 alleles/sample were studied at six microsatellite loci. Mutant fragments were identified, quantified, and compared using Poisson statistics. Most of the variants were small deletions or insertions, with more mutants being deletions, as has been previously described in yeast and transgenic mice. SP-PCR, where most of the pools contained only 3 or less fragments, enabled identification of variants too infrequent to be detected by large pool PCR. Mutant fragments in positive control MSI-H tumor samples ranged from 0.26 to 0.68 in at least 4 of the 6 loci tested and were consistent with their MSI-H status. In the so called MSS tumors and constitutive tissues (normal colon tissue, and PBLs) of all the HNPCC patients, low, but significant levels of MSI were seen in at least two of the loci studied. This phenomenon was not seen in the sporadic MSI constitutive tissues nor the normal controls and suggests haploinsufficiency, gain-of-function, or a dominant/negative basis of the instability in HNPCC patients carrying germline mutations for tumor suppressor genes. A different frequency and spectrum of mutant fragments suggests a different genetic basis (other than a major mutation in MLH1 or MSH2) for disease in MSI-L and MSS HNPCC patients. ^

Whole Genome Analysis of Pinkeye Scores in Angus Cattle

Infectious Bovine Keratoconjunctivitis (IBK), known as pinkeye, is a common infectious disease affecting the eyes of cattle. It is characterized by excessive tearing, inflammation of the conjunctiva, and ulceration of the cornea. Although pinkeye is non-fatal, it has a marked economic impact on the cattle industry, due to the decreased performance of infected individuals. Genetic effects on the susceptibility of IBK have been studied and Hereford, Jersey, and Holstein breeds were found to be more susceptible to IBK than Bos Indicus breeds. The objectives of our study were: 1) to estimate genetic parameters of IBK scored in different categories by using genomic threshold model, and 2) to detect markers in linkage disequilibrium with quantitative tract loci (QTL) associated with IBK.