Doctor of Philosophy

dissertationAdvances in technology have produced efficient and powerful scientific instruments for measuring biological phenomena. In particular, modern microscopes and nextgeneration sequencing machines produce data at such a rate that manual analysis is no longer practical or feasible for meaningful scientific inquiries. Thus, there is a great need for computational strategies to organize and analyze huge amounts of data produced by biological experiments. My work presents computational strategies and software solutions for application in image analysis, human variant prioritization, and metagenomics. The information content of images can be leveraged to answer an extremely broad spectrum of questions ranging from inquiries about basic biological processes to highly specific, application-driven inquiries like the efficacy of a pharmaceutical drug. Modern microscopes can produce images at a rate at which rigorous manual analysis is impossible. I have created software pipelines that automate image analysis in two specific applications domains. In addition, I discuss general image analysis strategies that can be applied to a wide variety of problems. There are tens of millions of known human genetic variants. Prioritizing human variants based on how likely they are to cause disease is of huge importance because of the potential impact on human health. Current variant prioritization methods are limited by their scope, efficiency, and accuracy. I present a variant prioritization method, the VAAST variant prioritizer, which is superior in its scope, efficiency, and accuracy to existing variant prioritization methods. The rise of next-generation sequencing enables huge quantities of sequence to be generated in a short period of time. No field of study has been affected by rapid sequencing more than metagenomics. Metagenomics, the genomic analysis of a population v of microorganisms, has important implications for pathogen detection because metagenomics enables the culture-free detection of microorganisms. I have created Taxonomer, a comprehensive metagenomics pipeline that enables the real-time analysis of read datasets derived from environmental samples

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA

5′UTR Variants of Ribosomal Protein S19 Transcript Determine Translational Efficiency: Implications for Diamond-Blackfan Anemia and Tissue Variability

Background: Diamond-Blackfan anemia (DBA) is a lineage specific and congenital erythroblastopenia. The disease is associated with mutations in genes encoding ribosomal proteins resulting in perturbed ribosomal subunit biosynthesis. The RPS19 gene is mutated in approximately 25 % of DBA patients and a variety of coding mutations have been described, all presumably leading to haploinsufficiency. A subset of patients carries rare polymorphic sequence variants within the 59untranslated region (59UTR) of RPS19. The functional significance of these variants remains unclear. Methodology/Principal Findings: We analyzed the distribution of transcriptional start sites (TSS) for RPS19 mRNAs in testis and K562 cells. Twenty-nine novel RPS19 transcripts were identified with different 59UTR length. Quantification of expressed w.t. 59UTR variants revealed that a short 59UTR correlates with high levels of RPS19. The total levels of RPS19 transcripts showed a broad variation between tissues. We also expressed three polymorphic RPS19 59UTR variants identified in DBA patients. The sequence variants include two insertions (c.-147_-146insGCCA and c.-147_-146insAGCC) and one deletion (c.-144_-141delTTTC). The three 59UTR polymorphisms are associated with a 20–30 % reduction in RPS19 protein levels when compared to the wild-type (w.t.) 59UTR of corresponding length. Conclusions: The RPS19 gene uses a broad range of TSS and a short 59UTR is associated with increased levels of RPS19. Comparisons between tissues showed a broad variation in the total amount of RPS19 mRNA and in the distribution of TS

Transiently Active Wnt/β-Catenin Signaling Is Not Required but Must Be Silenced for Stem Cell Function during Muscle Regeneration

Adult muscle’s exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/β-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/β-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated β-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/β-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which β-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/β-catenin signaling that is important for muscle regeneration

Human RPS19, the gene mutated in Diamond-Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits

Diamond-Blackfan anemia (DBA) typically presents with red blood cell aplasia that usually manifests in the first year of life. The only gene currently known to be mutated in DBA encodes ribosomal protein S19 (RPS19). Previous studies have shown that the yeast RPS19 protein is required for a specific step in the maturation of 40S ribosomal subunits. Our objective here was to determine whether the human RPS19 protein functions at a similar step in 40S subunit maturation. Studies where RPS19 expression is reduced by siRNA in the hematopoietic cell line, TF-1, show that human RPS19 is also required for a specific step in the maturation of 40S ribosomal subunits. This maturation defect can be monitored by studying rRNA-processing intermediates along the ribosome synthesis pathway. Analysis of these intermediates in CD34(−) cells from the bone marrow of patients with DBA harboring mutations in RPS19 revealed a pre-rRNA–processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34(+) cells from patients with DBA who have mutations in RPS19

The VAAST Variant Prioritizer (VVP): ultrafast, easy to use whole genome variant prioritization tool

Abstract Background Prioritization of sequence variants for diagnosis and discovery of Mendelian diseases is challenging, especially in large collections of whole genome sequences (WGS). Fast, scalable solutions are needed for discovery research, for clinical applications, and for curation of massive public variant repositories such as dbSNP and gnomAD. In response, we have developed VVP, the VAAST Variant Prioritizer. VVP is ultrafast, scales to even the largest variant repositories and genome collections, and its outputs are designed to simplify clinical interpretation of variants of uncertain significance. Results We show that scoring the entire contents of dbSNP (> 155 million variants) requires only 95 min using a machine with 4 cpus and 16 GB of RAM, and that a 60X WGS can be processed in less than 5 min. We also demonstrate that VVP can score variants anywhere in the genome, regardless of type, effect, or location. It does so by integrating sequence conservation, the type of sequence change, allele frequencies, variant burden, and zygosity. Finally, we also show that VVP scores are consistently accurate, and easily interpreted, traits not shared by many commonly used tools such as SIFT and CADD. Conclusions VVP provides rapid and scalable means to prioritize any sequence variant, anywhere in the genome, and its scores are designed to facilitate variant interpretation using ACMG and NHS guidelines. These traits make it well suited for operation on very large collections of WGS sequences

Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging since the phenotype is highly dependent on the level of RPS19 downregulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded downregulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene

Additional file 5: of The VAAST Variant Prioritizer (VVP): ultrafast, easy to use whole genome variant prioritization tool

Figure S5. VVP box plots for all ClinVar and NA12878 variants broken down by CADD scoring class. Note that in contrast to CADD’s scores for these same variants (see Additional file 4: Figure S4), VVP assigns high scores to FRAME_SHIFT and STOP_GAINED variants in ClinVar, but low scores for those same classes in NA12878. ClinVar scored as in Fig. 2a. NA12878 was scored using the observed zygosity of each variant. Score > 56 is threshold for damaging. (PDF 177 kb

Additional file 4: of The VAAST Variant Prioritizer (VVP): ultrafast, easy to use whole genome variant prioritization tool

Figure S4. CADD box plots for all ClinVar and NA12878 variants broken down by CADD scoring class. These results help to explain CADD’s call rate on NA12878. Note that CADD assigns high scores to FRAME_SHIFT and STOP_GAINED variants in both ClinVar and NA12878. Score > 23 is threshold for damaging. (PDF 152 kb

Additional file 2: of The VAAST Variant Prioritizer (VVP): ultrafast, easy to use whole genome variant prioritization tool

Figure S2. Violin plots for the ClinVar dataset. Scores have been normalized as in Fig. 3. Note how the VVP benign and pathogenic scores are better separated. (PDF 157 kb