Browsing by Subject "next-generation sequencing"

Genetics account for a large, mostly unexplained proportion of human disease. Though the role of genetics in simple, Mendelian traits has long been established, it is more difficult to disambiguate the role of various human genetic factors in complex disease traits. However, as genetics technology and methodology has advanced, from genome-wide association studies (GWAS) to next-generation sequencing (NGS), our ability to detect the role of both rare and common human genetic variation in complex disease traits has greatly improved, allowing us to demonstrate robust genetic factors involved in a variety of disease from metabolic to viral. However, despite the outstanding progress in human genetics, many complex disease traits lack robustly associated genetic variants, the existing variation only accounts for a small proportion of the estimated heritability, or the trait lacks comprehensive genetic investigation all together.

In this thesis I conducted a common variant study using GWAS and a comprehensive NGS analysis - both standards in the field - to investigate the role of human genetics in the severity of complex disease traits ranging from viral disease to metabolic: herpes simplex virus type 2 (HSV-2) and non-alcoholic fatty liver disease (NAFLD). Chapter 1 provides a broad overview of current human genetics methodologies and the advantages and caveats to each technology for complex disease traits, as well as the background and current state of genetics research for the two complex traits investigated: HSV-2 and NAFLD.

Chapter 2 utilizes a GWAS to investigate the role of common human genetic variation in HSV-2 severity, which has previously only been investigated through a small handful of candidate gene studies. We were unable to replicate previous candidate gene associations, though we did detect several variants in or near biologically plausible genes (including ABCA1 and KIF1B) that approached, though did not reach, genome-wide statistical significance with HSV-2 severity as measured by the quantitative viral shedding rate. This is the first genome-wide investigation of human genetics in HSV-2.

Chapter 3 utilizes whole-exome sequencing at both the single-variant and gene levels to further elucidate the role of human genetics in gold standard liver biopsy confirmed NAFLD fibrosis extreme phenotypes: protective and progressor. We were able to replicate known associations with PNPLA3 and TM6SF2 and advanced fibrosis, despite the limited available sample size. We also observed enrichment of variation in distinct genes for progressor or protective NAFLD phenotypes, though these genes did not reach statistical significance. This is the first NGS study of NAFLD, and thus the first investigation of the role of rare variation in NAFLD.

Overall, this thesis applied genome-wide techniques to interrogate gaps in the genetics of complex trait severity, from viral to liver disease, using unique, well-phenotyped cohorts. Human genetics remains a complicated field that will require the continued use of well-phenotyped cohorts in larger numbers, as well as both complementary and confirmatory sequencing and bioinformatics methods to fully detangle. While the research in this thesis is primarily hypothesis generating, and potentially associated variants will have to be replicated and investigated on a functional level to be confirmed as causal, the exploration of genetic associations with complex disease traits can prove highly informative for both understanding the underlying biology of these traits and for identifying genes and pathways that may act as biomarkers or treatment targets. Thus, this thesis has acted as a primer to expand knowledge of the role of human genetics in two highly complex and varied traits, HSV-2 and NAFLD, paving the way for further studies, ultimately with the goal of improving human health.

Neurodevelopmental disorders develop over time and are characterized by a wide variety of mental, behavioral, and physical phenotypes. The categorization of neurodevelopmental disorders encompasses a broad range of conditions including intellectual disability, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy, schizophrenia, bipolar disorder, and epilepsy, among others. Diagnostic classifications of neurodevelopmental disorders are complicated by comorbidities among these neurodevelopmental disorders, unidentified causal genes, and growing evidence of shared genetic risk factors.

We sought to identify the genetic underpinnings of a variety of neurodevelopmental disorders, with a particular emphasis on the epilepsies, by employing next–generation sequencing to thoroughly interrogate genetic variation in the human genome/exome. First, we investigated four families presenting with a seemingly identical and previously undescribed neurodevelopmental disorder characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. These families all exhibited an apparent autosomal recessive pattern of inheritance. Second, we investigated a heterogeneous cohort of ∼60 undiagnosed patients, the majority of whom suffered from severe neurodevelopmental disorders with a suspected genetic etiology. Third, we investigated 264 patients with epileptic encephalopathies — severe childhood epilepsy disorders — looking specifically at infantile spasms and Lennox–Gastaut syndrome. Finally, we investigated ∼40 large multiplex epilepsy families with complex phenotypic constellations and unclear modes of inheritance. The studied neurodevelopmental disorders exhibited a range of genetic complexity, from clear Mendelian disorders to common complex disorders, resulting in varying degrees of success in the identification of clearly causal genetic variants.

In the first project, we successfully identified the disease–causing gene. We show that recessive mutations in ASNS (encoding asparagine synthetase) are responsible for this previously undescribed neurodevelopmental disorder. We also characterized the causal mutations in vitro and studied Asns–deficient mice that mimicked aspects of the patient phenotype. This work describes ASNS deficiency as a novel neurodevelopmental disorder, identifies three distinct causal mutations in the ASNS gene, and indicates that asparagine synthesis is essential for the proper development and function of the brain.

In the second project, we exome sequenced 62 undiagnosed patients and their unaffected biological parents (trios). By analyzing all identified variants that were annotated as putatively functional and observed as a novel genotype in the probands (not observed in the unaffected parents or controls), we obtained a genetic diagnosis for 32% (20/62) of these patients. Additionally, we identify strong candidate variants in 31% (13/42) of the undiagnosed cases. We also present additional analysis methods for moving beyond traditional screens, e.g., considering only securely implicated genes, or subjecting qualifying variants from any gene to two unique analysis approaches. This work adds to the growing evidence for the utility of diagnostic exome sequencing, increases patient sizes for rare neurodevelopmental disorders (enabling more detailed analyses of the phenotypic spectrum), and proposes novel analysis approaches which will likely become beneficial as the number of sequenced undiagnosed patients grows.

In the third project, we again employ a trio–based exome sequencing design to investigate the role of de novo mutations in two classical forms of epileptic encephalopathy. We find a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 x 10–3, likelihood analysis). We provide clear statistical evidence for two novel genes associated with epileptic encephalopathy — GABRB3 and ALG13. Together with the 15 well–established epileptic encephalopathy genes, we statistically confirm the association of an additional ten putative epileptic encephalopathy genes. We show that only ∼12% of epileptic encephalopathy patients in our cohort are explained by de novo mutations in one of these 24 genes, highlighting the extreme locus heterogeneity of the epileptic encephalopathies.

Finally, we investigated multiplex epilepsy families to uncover novel epilepsy susceptibility factors. Candidate variants emerging from sequencing within discovery families were further assessed by cosegregation testing, variant association testing in a case–control cohort, and gene–based resequencing in a cohort of additional multiplex epilepsy families. Despite employing multiple approaches, we did not identify any clear genetic associations with epilepsy. This work has, however, identified a set of candidates that may include real risk factors for epilepsy; the most promising of these is the MYCBP2 gene. This work emphasizes the extremely high locus and allelic heterogeneity of the epilepsies and demonstrates that very large sample sizes are needed to uncover novel genetic risk factors.

Collectively, this body of work has securely implicated three novel neurodevelopmental disease genes that inform the underlying pathology of these disorders. Furthermore, in the final three studies, this work has highlighted additional candidate variants and genes that may ultimately be validated as disease–causing as sample sizes increase.