Genotypic spectrum and genotype-phenotype correlation of trimethylaminuria

Trimethylaminuria (TMAU) or Fish odor syndrome is an autosomal recessive disease that is characterized by pungent body odor with subsequent psychosocial complications. There are limited studies of the sequence variants causing TMAU in the literature with most studies describing only one or two patients and lacking genotype-phenotype correlations. Also to date, there is no laboratory in the US or Europe that offers TMA genetic testing on a clinical basis. We have recently validated genetic testing in the University of Colorado DNA Diagnostic Laboratory. We have a database of a few dozen patients with a biochemical diagnosis of TMA at the University of Colorado at Denver Health Sciences Center (UCDHSC) which includes a few patients with the classical form of the disease. We have used the newly established clinical test in our institution to attempt to characterize the genotype (sequence variants including mutations and polymorphisms) of classical TMAU patients and to establish a genotype-phenotype (biochemical and clinical) association. The questionnaire results confirmed most of the previously reported epidemiological findings of TMAU and also indicated that TMAU patients use multiple intervention measures in attempt to control their symptoms with dietary control being most effective. Despite the complexity of intervention, most patients did not have any medical follow up and there was underutilization of specialist care. In a set of our patients, two deleterious mutations were identified in 4/12 patients including a novel T237P sequence variant, while the majority of our patients (8/12) did not reveal any mutations. Some of the latter were double heterozygous for the E158K and E308G polymorphisms which could explain a mild phenotype while others had only the E158K variant which raised the question of undetected mutations. These results indicate that further experiments are needed to further delineate the full mutational spectrum of the FMO3 gene. ^

Polycomb repressive complex 2 and induced basal-like breast cancer phenotype mediated by cyclin E/Cdk2

Despite of much success of breast cancer treatment, basal-like breast cancer subtype still presented as a clinical challenge to mammary oncologist for its lack of available targeted therapy owing to their negative expression of targeted molecules, such as PgR, ERα and Her2. These molecules are all critical regulators in mammary gland development. EZH2, a histone methyltransferase, by forming Polycomb Repressive Complex 2(PRC2) can directly suppress a large array of developmental regulators. Overexpression of cyclin E has also been correlated with basal-like (triple-negative) breast cancer and poor prognosis. We found an important functional link between these two molecules. Cyclin E/Cdk2 can enhance PRC2 function by phosphorylating a specific residue of EZH2, threonine 416 and increasing EZH2's ability to complex with SUZ12. This regulation would further recruit whole PRC2 complex to core promoter regions of these developmental regulators. The local enrichment of PRC2 complex would then trimethylate H3K27 around the core promoter regions and suppress the expression of targeted genes, which included PgR, ERα, erbB2 and BRCA1. This widespread gene suppressive effect imposed by highly active PRC2 complex would then transform the lumina) type cell to adopt a basal-like phenotype. This finding suggested deregulated Cdk2 activity owing to cyclin E overexpression may contribute to basal phenotype through enhancing epigenetic silencing effects by regulating PRC2 function. Inhibition of Cdk2 activity in basal-like cancer cells may help release the suppression, reexpress the silenced genes and become responsive to existing anti-hormone or anti-Her2 therapy. From this study, the mechanisms described here provided a rationale to target basal-like breast cancer by new combinational therapy of Cdk2 inhibitors together with Lapatinib, or Aromatin. ^

Functional data analysis approaches for genotype-phenotype association studies from next-generation sequencing

Next-generation DNA sequencing platforms can effectively detect the entire spectrum of genomic variation and is emerging to be a major tool for systematic exploration of the universe of variants and interactions in the entire genome. However, the data produced by next-generation sequencing technologies will suffer from three basic problems: sequence errors, assembly errors, and missing data. Current statistical methods for genetic analysis are well suited for detecting the association of common variants, but are less suitable to rare variants. This raises great challenge for sequence-based genetic studies of complex diseases.^ This research dissertation utilized genome continuum model as a general principle, and stochastic calculus and functional data analysis as tools for developing novel and powerful statistical methods for next generation of association studies of both qualitative and quantitative traits in the context of sequencing data, which finally lead to shifting the paradigm of association analysis from the current locus-by-locus analysis to collectively analyzing genome regions.^ In this project, the functional principal component (FPC) methods coupled with high-dimensional data reduction techniques will be used to develop novel and powerful methods for testing the associations of the entire spectrum of genetic variation within a segment of genome or a gene regardless of whether the variants are common or rare.^ The classical quantitative genetics suffer from high type I error rates and low power for rare variants. To overcome these limitations for resequencing data, this project used functional linear models with scalar response to develop statistics for identifying quantitative trait loci (QTLs) for both common and rare variants. To illustrate their applications, the functional linear models were applied to five quantitative traits in Framingham heart studies. ^ This project proposed a novel concept of gene-gene co-association in which a gene or a genomic region is taken as a unit of association analysis and used stochastic calculus to develop a unified framework for testing the association of multiple genes or genomic regions for both common and rare alleles. The proposed methods were applied to gene-gene co-association analysis of psoriasis in two independent GWAS datasets which led to discovery of networks significantly associated with psoriasis.^

EFFECTS OF THE ACTA2 R258C MUTATION ON VASCULAR SMOOTH MUSCLE CELL PHENOTYPE AND PROPERTIES

Thoracic Aortic Aneurysms and Dissections (TAAD) are the fifteenth leading cause of death in the United States. About 15% of TAAD patients have family history of the disease. The most commonly mutated gene in these families is ACTA2, encoding smooth muscle-specific α-actin. ACTA2 missense mutations predispose individuals both to TAAD and to vascular occlusive disease of small, muscular arteries. Mice carrying an Acta2 R258C mutant transgene with a wildtype Acta2 promoter were generated and bred with Acta2-/- mice to decrease the wildtype: mutant Acta2 ratio. Acta2+/+ R258C TGmice have decreased aortic contractility without aortic disease. Acta2+/- R258C TG mice, however, have significant aortic dilatations by 12 weeks of age and a hyperproliferative response to injury. We characterized smooth muscle cells (SMCs) from bothmouse models under the hypothesis that mutant α-actin has a dominant negative effect, leading to impaired contractile filament formation/stability, improper focal adhesion maturation and increased proliferation. Explanted aortic SMCs from Acta2+/+ R258C TG mice are differentiated - they form intact filaments, express higher levels of contractile markers compared to wildtype SMCs and have predominantly nuclear Myocardin-Related Transcription Factor A (MRTF-A) localization. However, ultracentrifugation assays showed large unpolymerized actin fractions, suggesting that the filaments are brittle. In contrast, Acta2+/- R258C TG SMCs are less well-differentiated, with pools of unpolymerized actin, more cytoplasmic MRTF-A and decreased contractile protein expression compared to wildtype cells. Ultracentrifugation assays after treating Acta2+/- R258C TGSMCs with phalloidin showed actin filament fractions, indicating that mutant α-actin can polymerize into filaments. Both Acta2+/+ R258C TGand Acta2+/- R258C TGSMCs have larger and more peripheral focal adhesions compared to wildtype SMCs. Rac1 was more activated in Acta2+/+ R258C TGSMCs; both Rac1 and RhoA were less activated in Acta2+/- R258C TG SMCs, and FAK was more activated in both transgenic SMC lines compared to wildtype. Proliferation in both cell lines was significantly increased compared to wildtype cells and could be partially attenuated by inhibition of FAK or PDGFRβ. These data support a dominant negative effect of the Acta2 R258C mutation on the SMC phenotype, with increasing phenotypic severity when wildtype: mutant α-actin levels are decreased.

DETERMINING THE GENOTYPE-PHENOTYPE CONNECTION IN SYNTHETIC INDUCIBLE GENE EXPRESSION SYSTEMS

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise. Methods I created probabilistic biochemical reaction models from known behaviors of the tetR and rtTA genes, gene products, and their gene architectures. I then simplified these models to account for essential behaviors of gene expression systems. Finally, I used these models to predict behaviors of modified gene expression systems, which were experimentally verified. Results Cell growth, which is often ignored when formulating chemical kinetics models, was essential for understanding gene expression behavior. Models incorporating growth effects were used to explain unexpected reductions in gene expression noise, design a set of gene expression systems with “linear” dose-responses, and quantify the speed with which cells explored their fitness landscapes due to noisy gene expression. Conclusions Models incorporating noisy gene expression and cell division were necessary to design, understand, and predict the behaviors of synthetic gene expression systems. The methods and models developed here will allow investigators to more efficiently design new gene expression systems, and infer gene expression properties of TetR based systems.

Galectin-3 Enhances the Malignant Melanoma Phenotype by Regulating Autotaxin

In melanoma patient specimens and cell lines, the over expression of galectin-3 is associated with disease progression and metastatic potential. Herein, we have sought out to determine whether galectin-3 affects the malignant melanoma phenotype by regulating downstream target genes. To that end, galectin-3 was stably silenced by utilizing the lentivirus-incorporated small hairpin RNA in two metastatic melanoma cell lines, WM2664 and A375SM, and subjected to gene expression microarray analysis. We identified and validated the lysophospholipase D enzyme, autotaxin, a promoter of migration, invasion, and tumorigenesis, to be down regulated after silencing galectin-3. Silencing galectin-3 significantly reduced the promoter activity of autotaxin. Interestingly, we also found the transcription factor NFAT1 to have reduced protein expression after silencing galectin-3. Electrophoretic mobility shift assays from previous reports have shown that NFAT1 binds to the autotaxin promoter in two locations. ChIP analysis was performed, and we observed a complete loss of bound NFAT1 to the autotaxin promoter after silencing galectin-3 in melanoma cells. Mutation of the NFAT1 binding sites at either location reduces autotaxin promoter activity. Silencing NFAT1 reduces autotaxin expression while over expressing NFAT1 in NFAT1 negative SB-2 melanoma cells induces autotaxin expression. These data suggest that galectin-3 silencing reduces autotaxin transcription by reducing the amount of NFAT1 protein expression. Rescue of galectin-3 rescues both NFAT1 and autotaxin. We also show that the re-expression of autotaxin in galectin-3 shRNA melanoma cells rescues the angiogenic phenotype in vivo. Furthermore, we identify NFAT1 as a potent inducer of tumor growth and experimental lung metastasis. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin and assign a novel role for NFAT1 during melanoma progression.

RNA-SEQUENCING APPLICATIONS: GENE EXPRESSION QUANTIFICATION AND METHYLATOR PHENOTYPE IDENTIFICATION

My dissertation focuses on two aspects of RNA sequencing technology. The first is the methodology for modeling the overdispersion inherent in RNA-seq data for differential expression analysis. This aspect is addressed in three sections. The second aspect is the application of RNA-seq data to identify the CpG island methylator phenotype (CIMP) by integrating datasets of mRNA expression level and DNA methylation status. Section 1: The cost of DNA sequencing has reduced dramatically in the past decade. Consequently, genomic research increasingly depends on sequencing technology. However it remains elusive how the sequencing capacity influences the accuracy of mRNA expression measurement. We observe that accuracy improves along with the increasing sequencing depth. To model the overdispersion, we use the beta-binomial distribution with a new parameter indicating the dependency between overdispersion and sequencing depth. Our modified beta-binomial model performs better than the binomial or the pure beta-binomial model with a lower false discovery rate. Section 2: Although a number of methods have been proposed in order to accurately analyze differential RNA expression on the gene level, modeling on the base pair level is required. Here, we find that the overdispersion rate decreases as the sequencing depth increases on the base pair level. Also, we propose four models and compare them with each other. As expected, our beta binomial model with a dynamic overdispersion rate is shown to be superior. Section 3: We investigate biases in RNA-seq by exploring the measurement of the external control, spike-in RNA. This study is based on two datasets with spike-in controls obtained from a recent study. We observe an undiscovered bias in the measurement of the spike-in transcripts that arises from the influence of the sample transcripts in RNA-seq. Also, we find that this influence is related to the local sequence of the random hexamer that is used in priming. We suggest a model of the inequality between samples and to correct this type of bias. Section 4: The expression of a gene can be turned off when its promoter is highly methylated. Several studies have reported that a clear threshold effect exists in gene silencing that is mediated by DNA methylation. It is reasonable to assume the thresholds are specific for each gene. It is also intriguing to investigate genes that are largely controlled by DNA methylation. These genes are called “L-shaped” genes. We develop a method to determine the DNA methylation threshold and identify a new CIMP of BRCA. In conclusion, we provide a detailed understanding of the relationship between the overdispersion rate and sequencing depth. And we reveal a new bias in RNA-seq and provide a detailed understanding of the relationship between this new bias and the local sequence. Also we develop a powerful method to dichotomize methylation status and consequently we identify a new CIMP of breast cancer with a distinct classification of molecular characteristics and clinical features.

Altered tumorigenic phenotype in mice with a hypomorphic p53 mutant allele

Mutations in the p53 tumor suppressor gene are found in over 50% of human tumors and in the germline of Li-Fraumeni syndrome families. About 80% of these mutations are missense in nature. In order to study how p53 missense mutations affect tumorigenesis in vivo, we focused on the murine p53 arg-to-his mutation at amino acid 172, which corresponds to the human hot spot mutation at amino acid 175. The double replacement procedure was employed to introduce the p53 R172H mutation into the p53 locus of ES cells and mice were generated. An additional 1bp deletion in the intron 2 splice acceptor site was detected in the same allele in mice. We named this allele p53R172HΔg. This allele makes a small amount of full length p53 mutant protein. ^ Spontaneous tumor formation and survival were studied in these mice. Mice heterozygous for the p53R172HΔg allele showed 50% survival at 17 months of age, similar to the p53+/− mice. Moreover, the p53R172HΔg/+ mice showed a distinct tumor spectrum: 55% sarcomas, including osteosarcoms, fibrosarcomas and angiosarcomas; 27% carcinomas, including lung adenocarcinomas, squamous cell carcinomas, hepatocellular carcinomas and islet cell carcinomas; and 18% lymphomas. Compared to the p53+/− mice, there was a clear increase in the frequency of carcinoma development and a decrease in lymphoma incidence. Among the sarcomas that developed, fibrosarcomas in the skin were also more frequently observed. More importantly, osteosarcomas and carinomas that developed in the p53R172HΔg/+ mice metastasized at very high frequency (64% and 67%, respectively) compared with less than 10% in the p53+/− mice. The metastatic lesions were usually found in lung and liver, and less frequently in other tissues. The altered tumor spectrum in the mice and increased metastatic potential of the tumors suggested that the p53R172H mutation represents a gain-of-function. ^ Mouse embryonic fibroblasts (MEFs) from the mice homozygous and heterozygous for the p53R172HΔg allele were studied for growth characteristics, immortalization potential and genomic instability. All of the p53R172HΔg /+ MEF lines are immortalized under a 3T3 protocol while under the same protocol p53+/− MEFs are not immortalized. Karyotype analysis showed a persistent appearance of chromosome end-to-end fusion in the MEFs both homozygous and heterozygous for the p53R172HΔg allele. These observations suggest that increased genomic instability in the cells may cause the altered tumor phenotypes. ^

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

A phenotype-based screen for embryonic lethal mutations in the mouse

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-γ, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a “forbidden” cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line and vice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cells in vivo.

Overexpression of DNA polymerase β in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs

DNA polymerase β (pol β) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol β cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol β. In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol β overexpression in cancer predisposition and tumor progression during chemotherapy.

Genetic and root phenotype diversity in Sri Lankan rice landraces may be related to drought resistance

Acknowledgements Mayuri Munasinghe was supported by a Commonwealth Scholarship (ref no. LKCS-2009-384). The development and use of the SNP chip was funded by a BBSRC grant BB/J003336/1. The authors thank Owen Price (University of Wollongong, Australia) for producing the coloured province map of Sri Lanka, Gareth Norton (Aberdeen) for merging the RDP1 SNP data with the Sri Lankan data and Tony Travis (Aberdeen) for help with PCA.

Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.