Browsing by Author "Marchuk, Douglas A"
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Item Open Access A cross-species approach using an in vivo evaluation platform in mice demonstrates that sequence variation in human RABEP2 modulates ischemic stroke outcomes.(American journal of human genetics, 2022-10) Lee, Han Kyu; Kwon, Do Hoon; Aylor, David L; Marchuk, Douglas AIschemic stroke, caused by vessel blockage, results in cerebral infarction, the death of brain tissue. Previously, quantitative trait locus (QTL) mapping of cerebral infarct volume and collateral vessel number identified a single, strong genetic locus regulating both phenotypes. Additional studies identified RAB GTPase-binding effector protein 2 (Rabep2) as the casual gene. However, there is yet no evidence that variation in the human ortholog of this gene plays any role in ischemic stroke outcomes. We established an in vivo evaluation platform in mice by using adeno-associated virus (AAV) gene replacement and verified that both mouse and human RABEP2 rescue the mouse Rabep2 knockout ischemic stroke volume and collateral vessel phenotypes. Importantly, this cross-species complementation enabled us to experimentally investigate the functional effects of coding sequence variation in human RABEP2. We chose four coding variants from the human population that are predicted by multiple in silico algorithms to be damaging to RABEP2 function. In vitro and in vivo analyses verify that all four led to decreased collateral vessel connections and increased infarct volume. Thus, there are naturally occurring loss-of-function alleles. This cross-species approach will expand the number of targets for therapeutics development for ischemic stroke.Item Open Access A Neuroprotective Locus Modulates Ischemic Stroke Infarction Independent of Collateral Vessel Anatomy.(Frontiers in neuroscience, 2021-01) Lee, Han Kyu; Wetzel-Strong, Sarah E; Aylor, David L; Marchuk, Douglas AAlthough studies with inbred strains of mice have shown that infarct size is largely determined by the extent of collateral vessel connections between arteries in the brain that enable reperfusion of the ischemic territory, we have identified strain pairs that do not vary in this vascular phenotype, but which nonetheless exhibit large differences in infarct size. In this study we performed quantitative trait locus (QTL) mapping in mice from an intercross between two such strains, WSB/EiJ (WSB) and C57BL/6J (B6). This QTL mapping revealed only one neuroprotective locus on Chromosome 8 (Chr 8) that co-localizes with a neuroprotective locus we mapped previously from F2 progeny between C3H/HeJ (C3H) and B6. The allele-specific phenotypic effect on infarct volume at the genetic region identified by these two independent mappings was in the opposite direction of the parental strain phenotype; namely, the B6 allele conferred increased susceptibility to ischemic infarction. Through two reciprocal congenic mouse lines with either the C3H or B6 background at the Chr 8 locus, we verified the neuroprotective effects of this genetic region that modulates infarct volume without any effect on the collateral vasculature. Additionally, we surveyed non-synonymous coding SNPs and performed RNA-sequencing analysis to identify potential candidate genes within the genetic interval. Through these approaches, we suggest new genes for future mechanistic studies of infarction following ischemic stroke, which may represent novel gene/protein targets for therapeutic development.Item Open Access Cerebral Cavernous Malformations: From Two-Hit Mechanism to Developing a Targeted Therapy(2013) McDonald, David AndrewCerebral cavernous malformations (CCMs) are multicavernous vascular lesions affecting the central nervous system. Affected individuals have a lifetime risk of recurrent headaches, focal neurological deficits, seizures, and intracerebral hemorrhage leading to stroke. Patients tend to fall into two classes: familial cases with a known family history and multiple lesions, and; sporadic cases with no family history and single lesions. This epidemiological pattern suggests a two-hit mutational mechanism for CCM. While somatic mutations have been identified in lesions from familial patients, it is unknown if sporadic cases follow the same genetic mechanism. Using a next-generation sequencing strategy, I have identified somatic mutations from sporadic CCM lesions in the three known CCM genes, including one lesion bearing two independent mutations in CCM1. These data support a two-hit mutation mechanism in CCM for sporadic patients.
The mechanism of CCM pathogenesis (how mutations in one of the three CCM genes causes lesions to form and develop) is currently unknown. We developed mouse models that recapitulate the human disease. We have further shown that inhibition of Rho Kinase decreases the number of late-stage, multicavernous lesions. This is the first potential therapeutic strategy to specifically treat CCM, and suggests that the RhoA pathway is a central player in CCM pathogenesis.
Item Open Access Functional Evaluation of Causal Mutations Identified in Human Genetic Studies(2016) Lu, Yi-FanHuman 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.
Item Open Access Genetic Modifiers in Response to Ischemia(2010) Keum, SehoonIn a mouse model of ischemic stroke, infarct volume is highly variable and strain dependent, but the natural genetic determinants responsible for this difference remain unknown. To identify genetic determinants regulating ischemic neuronal damage and to dissect apart the role of individual genes and physiological mechanisms in infarction in mice, we performed forward genetic mapping analyses of surgically induced cerebral infarct volume. We have identified multiple quantitative trait loci (QTL) that modulate infarct volume, with a major locus (Civq1 ) on chromosome 7 accounting for over 50% of the variation, with a combined LOD score of 21.7. Measurement of infarct volume in chromosome substitution strains (CSS) and two additional intercrosses validate that Civq1 on chromosome 7 is present in multiple inbred strains. Interval-specific ancestral SNP haplotype analysis for Civq1 results in 5 candidate genes. A causative gene underlying Civq1 may regulate collateral artery formation and genetic variations in the gene may result in the differential outcome of cerebral infarction. Additionally, we have identified a locus of large effect, Civq4, modulating infarct volume through a mechanism different from collateral circulation. In conclusion, the extent of ischemic tissue damage after distal middle cerebral artery occlusion (MCAO) in inbred strains of mice is regulated by genetic variation mapping to at least 4 different loci. A single locus on chromosome 7 determines the majority of the observed variation in the trait in multiple mouse strains. Civq1 appears to be identical to Lsq1, a locus conferring limb salvage and reperfusion in hindlimb ischemia. The identification of the genes underlying these loci may uncover novel genetic and physiological pathways that modulate cerebral infarction and provide new targets for therapeutic intervention in ischemic stroke, and possibly other human vascular occlusive diseases.
Item Open Access Human Genomics of Complex Trait Severity(2017) Kleinstein, Sarah ElizabethGenetics 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.
Item Open Access Inhibition of Nucleolar Proteins in Caenorhabditis Elegans Confers Enhanced Resistance to Salmonella Enterica through a P53/cep-1-Dependent Mechanism(2009) Fuhrman, Laura ElizabethThe relatively simple innate immune system of Caenorhabditis elegans and the number of traits that facilitate genetic and genomic analysis using this organism have nurtured rapid advances into the understanding of C. elegans innate immunity during the last few years. However, traditional methods of isolating and mapping C. elegans mutants exhibiting aberrant immune responses to pathogen infection are often labor intensive and time consuming. Therefore, a simple and rapid means of isolating and mapping C. elegans immune mutants will increase the number of mutants that can be studied. Salmonella enterica, as well as other bacterial pathogens, has been described to cause a significant distension of the C. elegans intestinal lumen, which correlates with death of the nematode. C. elegans mutants which exhibit a weakened immune response would therefore be expected to develop intestinal distension at an earlier time point than wild type. Likewise, mutants which exhibit an enhanced immune response would be expected to develop intestinal distension at a later time point than wild type. Taking advantage of this correlation, we designed a novel approach to isolating C. elegans mutants which exhibit aberrant immune responses to the bacterial pathogen, S. enterica. Furthermore, we validated and optimized the use of Amplifluor®, a high-throughput genotyping system, for use in C. elegans single nucleotide polymorphism (SNP) mapping.
To date, the only known negative regulators of innate immunity in C. elegans are dependent on the FOXO transcription factor, DAF-16 and regulate lifespan in addition to immunity. Therefore, we focused our efforts on identifying additional negative regulators of innate immunity by screening for mutants which display a reduced accumulation of S. enterica at a time point when wild-type nematodes are packed with bacteria. In a genetic screen for C. elegans mutants which display reduced accumulation of S. enterica/GFP, we identified a mutation in nol-6, a nucleolar protein containing a nucleolar RNA-associated protein (Nrap) domain which is conserved across eukaryotic organisms. nol-6 is implicated in ribosomal RNA (rRNA) processing during the early stages of ribosome biogenesis. We show that knockdown of nol-6 as well as other nucleolar genes leads to a reduction of pathogen accumulation and enhanced resistance to killing by pathogen. In addition, we demonstrate that enhanced resistance is dependent on p53/cep-1. Furthermore, microarray analysis shows a significant enrichment of upregulated genes that have previously been shown to be dependent on p53/cep-1 for induction following ultraviolet radiation. These results represent the first evidence that C. elegans innate immunity is regulated by the nucleolus through a p53/cep-1-dependent mechanism.
Item Open Access Investigating the Pathogenesis and Response to Therapy of Cerebral Cavernous Malformations Using Transgenic Murine Models(2021) Detter, Matthew RobertCerebral cavernous malformations (CCMs), also known as cavernous angiomas, are clusters of sinusoidal capillary-venous vessels that develop in the approximately 1 in 200 individuals. With a gross appearance likened to a mulberry, CCMs have a disrupted endothelial barrier that can lead to patients presenting with focal neurologic deficits, seizures, headaches, and recurrent hemorrhages. Current therapy for CCMs consists of surgical removal in select cases and symptom management. There is no medical therapy to treat the underlying pathology in this disease. CCMs develop following biallelic loss-of-function mutations in CCM1, CCM2, or CCM3. These malformations develop sporadically, often presenting as a solitary lesion, or in an autosomal dominant pattern of inheritance in which individuals develop 10 to 100s of CCMs. How a single mutant endothelial cell leads to the formation of large, multicellular malformation is not known. My doctoral work utilized transgenic murine models of CCM to investigate 1) the early events of CCM formation and 2) the ability of proposed drugs to treat CCMs. We discovered that mutant endothelial cells undergo clonal expansion and incorporate wild-type endothelial cells as the malformation grows. We also developed novel murine models that recapitulate the chronic and acute CCM hemorrhage seen in patients. These new insights and transgenic tools further our understanding of this disease and advance the research community’s efforts to identify a medical treatment for CCMs.
Item Open Access INVESTIGATION OF GENETIC FACTORS DETERMINING ISCHEMIC STROKE OUTCOME(2013) Chu, PeiLunCerebrovascular disease (stroke), especially ischemic stroke, is a major cause of death and neurological disability in adults. Because of its clinical heterogeneity, stroke is considered as a multi-factorial and polygenic disorder. Most current genetic studies of ischemic stroke focus on genetic susceptibility rather than factors determining stroke outcome. The genetic components of ischemic stroke outcome are difficult to study in humans due to environmental factors and medical intervention. Thus, we proposed to use a surgically induced, permanent, focal cerebral ischemic stroke mouse model to investigate genetic factors of ischemic stroke outcome measured by infarct volume. This model is the middle cerebral artery occlusion (MCAO) model. First, we screened infarct volumes across 32 inbred mouse strains. The infarct volume varies between strains, and this strongly suggests that infarct volume is genetically determined. To identify these genetic factors, we used genome-wide association study [Efficient Mixed-Model Association (EMMA) analysis] on infarct volume from 32 inbred mouse strains. Using the EMMA analysis, we identified 11 infarct volume-associated loci; however, most loci were mapped with missing alleles. This suggests that these loci might be false positives. Thus, we used specifically designed scripts of EMMA analysis with updated mouse SNP database to correct for potential false positives. The loci identified by the updated EMMA analyses will led us to the identification of genes involved in ischemic stroke outcome.
There are two major mechanisms were proposed to be determinants of infarct volume, the extent of native collateral circulation and neuroprotection. Using the infarct volume screening panel from 32 inbred strains, we observed that infarct volume is inversely correlated with the native collateral vessel number. However, among these inbred strains, we also observed several strains differ significantly in infarct volumes but harbor similar collateral numbers. In order to identify genetic factors determining infarct volume in a collateral-independent manner (neuroprotection), we used quantitative trait locus (QTL) mapping on mouse strains that exhibit the most difference in infarct volumes but the least difference in collateral numbers (C57BL/6J and C3H/HeJ). From the F2 B6 x C3H cross, we mapped 4 loci determining infarct volume (cerebral infarct volume QTL 4 to 7, Civq4 to Civq7). The Civq4 locus is the strongest locus (LOD 9.8) that contributes 21% of phenotypic variance in infarct volume. We also used a parallel F2 B6 x C3H cross to perform a QTL mapping on collateral vessel traits to further verify these collateral-independent loci. Among these 4 loci, the Civq4 and Civq7 loci appear to be truly collateral-independent. Based on strain-specific sequence variants and mRNA expression differences, we proposed Msr1 and Mtmr7 are the potential candidate genes of the Civq4 locus. Identification of the collateral-independent genetic factors will help to understand the genetic architecture, disease pathophysiology and potential therapeutic targets for of ischemic stroke
Item Open Access Investigation of the Molecular Mechanism for Cerebral Cavernous Malformations(2009) Akers, Amy LeeCerebral cavernous malformations (CCM) are vascular anomalies of the central nervous system comprised of grossly-dilated blood-filled capillaries. CCM leisons may occur sporadically or by inheritance of a mutation in one of three genes, CCM1, CCM2, or CCM3. Prior to the identification of the genes involved in pathogenesis, sporadic and inherited cases could be distinguished by lesion burden where sporadic cases exclusively showed single lesions, and patients with inherited disease developed multiple lesions. This observation lead us to hypothesize that CCM lesion genesis may follow a two-hit genetic mechanism. To investigate this hypothesis and determine the molecular mechanism underlying CCM pathogeneis, we used resected human lesion samples to identify biallelic somatic and germline mutations that are specific to the lesion endothelium. Additionaly, we created mouse models in which heterozygosity of Ccm1 or Ccm2 in conjunction with deficiency for either the p53 or Msh2 genes, recapitulates the genetic and phenotypic properties of the human condition. In conclusion, we have provided evidence that CCM lesion genesis requires inactivation of both allelic copies for CCM1, CCM2, or CCM3within a subset of vascular endothelium.
Item Open Access Natural allelic variation of the IL-21 receptor modulates ischemic stroke infarct volume.(The Journal of clinical investigation, 2016-08) Lee, Han Kyu; Keum, Sehoon; Sheng, Huaxin; Warner, David S; Lo, Donald C; Marchuk, Douglas ARisk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.Item Open Access Natural genetic variation of integrin alpha L (Itgal) modulates ischemic brain injury in stroke.(PLoS genetics, 2013-01) Keum, Sehoon; Lee, Han Kyu; Chu, Pei-Lun; Kan, Matthew J; Huang, Min-Nung; Gallione, Carol J; Gunn, Michael D; Lo, Donald C; Marchuk, Douglas ADuring ischemic stroke, occlusion of the cerebrovasculature causes neuronal cell death (infarction), but naturally occurring genetic factors modulating infarction have been difficult to identify in human populations. In a surgically induced mouse model of ischemic stroke, we have previously mapped Civq1 to distal chromosome 7 as a quantitative trait locus determining infarct volume. In this study, genome-wide association mapping using 32 inbred mouse strains and an additional linkage scan for infarct volume confirmed that the size of the infarct is determined by ancestral alleles of the causative gene(s). The genetically isolated Civq1 locus in reciprocal recombinant congenic mice refined the critical interval and demonstrated that infarct size is determined by both vascular (collateral vessel anatomy) and non-vascular (neuroprotection) effects. Through the use of interval-specific SNP haplotype analysis, we further refined the Civq1 locus and identified integrin alpha L (Itgal) as one of the causative genes for Civq1. Itgal is the only gene that exhibits both strain-specific amino acid substitutions and expression differences. Coding SNPs, a 5-bp insertion in exon 30b, and increased mRNA and protein expression of a splice variant of the gene (Itgal-003, ENSMUST00000120857), all segregate with infarct volume. Mice lacking Itgal show increased neuronal cell death in both ex vivo brain slice and in vivo focal cerebral ischemia. Our data demonstrate that sequence variation in Itgal modulates ischemic brain injury, and that infarct volume is determined by both vascular and non-vascular mechanisms.Item Open Access Neuronal IL-4Rα modulates neuronal apoptosis and cell viability during the acute phases of cerebral ischemia.(The FEBS journal, 2018-08) Lee, Han Kyu; Koh, Sehwon; Lo, Donald C; Marchuk, Douglas AIschemic stroke caused by an embolus or local thrombosis results in neural tissue damage (an infarct) in the territory of the occluded cerebral artery. Decades of studies have increased our understanding of the molecular events during cerebral infarction; however, translation of these discoveries to druggable targets for ischemic stroke treatment has been largely disappointing. Interleukin-4 (IL-4) is a multifunctional cytokine that exerts its cellular activities via the interleukin-4 receptor α (IL-4Rα). This cytokine receptor complex is associated with diverse immune and inflammatory responses. Recent studies have suggested a role of the cytokine IL-4 in long-term ischemic stroke recovery, involving immune cell activity. In contrast, the role of the receptor, IL-4Rα especially in the acute phase of infarction is unclear. In this study, we determined that IL-4Rα is expressed on neurons and that during the early phases of cerebral infarction (24 h) levels of this receptor are increased to regulate cellular apoptosis factors through activation of STAT6. In this context, we show a neuroprotective role for IL-4Rα in an in vivo surgical model of cerebral ischemia and in ex vivo brain slice explants, using both genetic knockout of this receptor and RNAi-mediated gene knockdown. IL-4Rα may therefore represent a novel target and pathway for therapeutic development in ischemic stroke.Item Open Access Novel Neuroprotective Loci Modulating Ischemic Stroke Volume in Wild-Derived Inbred Mouse Strains.(Genetics, 2019-11) Lee, Han Kyu; Widmayer, Samuel J; Huang, Min-Nung; Aylor, David L; Marchuk, Douglas ATo identify genes involved in cerebral infarction, we have employed a forward genetic approach in inbred mouse strains, using quantitative trait loci (QTL) mapping for cerebral infarct volume after middle cerebral artery occlusion. We had previously observed that infarct volume is inversely correlated with cerebral collateral vessel density in most strains. In this study, we expanded the pool of allelic variation among classical inbred mouse strains by utilizing the eight founder strains of the Collaborative Cross and found a wild-derived strain, WSB/EiJ, that breaks this general rule that collateral vessel density inversely correlates with infarct volume. WSB/EiJ and another wild-derived strain, CAST/EiJ, show the highest collateral vessel densities of any inbred strain, but infarct volume of WSB/EiJ mice is 8.7-fold larger than that of CAST/EiJ mice. QTL mapping between these strains identified four new neuroprotective loci modulating cerebral infarct volume while not affecting collateral vessel phenotypes. To identify causative variants in genes, we surveyed nonsynonymous coding SNPs between CAST/EiJ and WSB/EiJ and found 96 genes harboring coding SNPs predicted to be damaging and mapping within one of the four intervals. In addition, we performed RNA-sequencing for brain tissue of CAST/EiJ and WSB/EiJ mice and identified 79 candidate genes mapping in one of the four intervals showing strain-specific differences in expression. The identification of the genes underlying these neuroprotective loci will provide new understanding of genetic risk factors of ischemic stroke, which may provide novel targets for future therapeutic intervention of human ischemic stroke.Item Open Access Patho-Genetic Characterization of the Muscular Dystrophy Gene Myotilin(2007-05-02T16:16:01Z) Garvey, Sean MichaelMyotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with Limb-Girdle Muscular Dystrophy Type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), and distal myopathy, four similar adult-onset, progressive, and autosomal dominant muscular dystrophies--collectively called the myotilinopathies. It is not yet known how myotilin mutations cause muscle disease. To investigate myotilin's role in the pathogenesis of muscle disease, I have created and characterized transgenic mice expressing mutant (Thr57Ile) myotilin under the control of the human skeletal alpha-actin promoter. Like LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization, and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. I show that the mutant myotilin protein properly localizes to the Z-disc, and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus (EDL) muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared to littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. To better understand myotilin function, I also created two separate lines of myotilin domain deletion transgenic mice: one expresses a deletion of the N-terminal domain and the second expresses a deletion of the minimal alpha-actinin binding site. Studies in these mice show that 1) the N-terminal domain of myotilin may be required for normal localization to the Z-disc; 2) interaction with alpha-actinin is not required for localization of myotilin to the Z-disc; and 3) deletion of the alpha-actinin binding site causes an aggregation phenotype similar to that of the TgT57I mouse and myotilinopathy patients. In sum, I have established a promising patho-physiological mouse model that unifies the diverse clinical phenotypes of the myotilinopathies. This mouse model promises to be a key resource for understanding myotilin function, unraveling LGMD1A pathogenesis, and investigating therapeutics.Item Open Access Somatic Mutations Drive Vascular Malformation Initiation, Progression, and Predisposition(2022) Snellings, DanielVascular malformations are a diverse class of focal lesions that may occur throughout the body and affect different vascular beds. Since the advent of next-generation sequencing it has become clear that virtually all vascular malformations are caused by postzygotic genetic changes occurring in a single cell: somatic mutations.Somatic mutations are widely accepted to be the initial, catalyzing event for vascular malformation. These mutations are a mix of gain-of-function and loss- of-function and occur in canonical vascular genes, or known oncogenes. From the past two decades of research, we have identified numerous genes which contribute to vascular malformation; yet, despite this genetic diversity, the mutations identified in individual malformations has thus far been—without exception—monogenic. In this document I present the first type of vascular malformation where digenic somatic mutations are a common and critical component of lesion pathogenesis. Furthermore, I propose that somatic mutations cause not only lesion initiation, but may drive the initiation, progression, and predisposition to vascular malformation. In support of this hypothesis, I first identify that vascular malformations in hereditary hemorrhagic telangiectasia are initiated by somatic mutations via a two- hit mechanism. Second, I determine that cerebral cavernous malformations harbor up to 3 distinct somatic mutations that synergize to fuel lesion progression. Finally, I show that developmental venous anomalies harbor a somatic mutation which creates a mosaic field of mutant cells that predisposes to cerebral cavernous malformations.