Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Genetic and Molecular Basis of Encapsulation and Capsule Diversity in Kingella kingae

Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation.

We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→.

To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→.

Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.

To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination.

In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.

The Roles of Phenotypic Plasticity and Plant-Microbe Interactions in the Evolution of Complex Traits in Boechera stricta

All organisms live in complex habitats that shape the course of their evolution by altering the phenotype expressed by a given genotype (a phenomenon known as phenotypic plasticity) and simultaneously by determining the evolutionary fitness of that phenotype. In some cases, phenotypic evolution may alter the environment experienced by future generations. This dissertation describes how genetic and environmental variation act synergistically to affect the evolution of glucosinolate defensive chemistry and flowering time in Boechera stricta, a wild perennial herb. I focus particularly on plant-associated microbes as a part of the plant’s environment that may alter trait evolution and in turn be affected by the evolution of those traits. In the first chapter I measure glucosinolate production and reproductive fitness of over 1,500 plants grown in common gardens in four diverse natural habitats, to describe how patterns of plasticity and natural selection intersect and may influence glucosinolate evolution. I detected extensive genetic variation for glucosinolate plasticity and determined that plasticity may aid colonization of new habitats by moving phenotypes in the same direction as natural selection. In the second chapter I conduct a greenhouse experiment to test whether naturally-occurring soil microbial communities contributed to the differences in phenotype and selection that I observed in the field experiment. I found that soil microbes cause plasticity of flowering time but not glucosinolate production, and that they may contribute to natural selection on both traits; thus, non-pathogenic plant-associated microbes are an environmental feature that could shape plant evolution. In the third chapter, I combine a multi-year, multi-habitat field experiment with high-throughput amplicon sequencing to determine whether B. stricta-associated microbial communities are shaped by plant genetic variation. I found that plant genotype predicts the diversity and composition of leaf-dwelling bacterial communities, but not root-associated bacterial communities. Furthermore, patterns of host genetic control over associated bacteria were largely site-dependent, indicating an important role for genotype-by-environment interactions in microbiome assembly. Together, my results suggest that soil microbes influence the evolution of plant functional traits and, because they are sensitive to plant genetic variation, this trait evolution may alter the microbial neighborhood of future B. stricta generations. Complex patterns of plasticity, selection, and symbiosis in natural habitats may impact the evolution of glucosinolate profiles in Boechera stricta.