ABSTRACT Variants in the LIM homeobox transcription factor 1-beta ( LMX1B ) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1b V265D (also known as Lmx1b Icst ) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA- Pde6b + /J (C3H) and DBA/2J- Gpnmb + (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1b V265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1b V265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1b V265D -induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b -induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments.

ABSTRACT Spinal muscular atrophy (SMA) is a devastating autosomal recessive neuromuscular disease resulting in muscle atrophy and neurodegeneration, and is the leading genetic cause of infant death. SMA arises when there are homozygous deletion mutations in the human SMN1 gene, leading to a decrease in corresponding SMN1 protein. Although SMN1 is expressed across multiple tissue types, much of the previous research into SMA focused on the neuronal aspect of the disease, overlooking many of the potential non-neuronal aspects of the disease. Therefore, we sought to address this gap in knowledge by modeling SMA in the nematode Caenorhabditis elegans . We mutated a previously uncharacterized allele, which resulted in the onset of mild SMA-like phenotypes, allowing us to monitor the onset of phenotypes at different stages. We observed that these mutant animals recapitulated many key features of the human disease, and most importantly, we observed that muscle dysfunction preceded neurodegeneration. Furthermore, we tested the therapeutic efficacy of targeting endoplasmic reticulum (ER) stress in non-neuronal cells and found it to be more effective than targeting ER stress in neuronal cells. We also found that the most potent therapeutic potential came from a combination of ER- and neuromuscular junction-targeted drugs. Together, our results suggest an important non-neuronal component of SMA pathology and highlight new considerations for therapeutic intervention.

ABSTRACT The global burden of neurodegenerative diseases underscores the urgent need for innovative strategies to define new drug targets and disease-modifying factors. The nematode Caenorhabditis elegans has served as the experimental subject for multiple transformative discoveries that have redefined our understanding of biology for ∼60 years. More recently, the considerable attributes of C. elegans have been applied to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease. Transgenic nematodes with genes encoding normal and disease variants of proteins at the single- or multi-copy level under neuronal-specific promoters limits expression to select neuronal subtypes. The anatomical transparency of C. elegans affords the use of co-expressed fluorescent proteins to follow the progression of neurodegeneration as the animals age. Significantly, a completely defined connectome facilitates detailed understanding of the impact of neurodegeneration on organismal health and offers a unique capacity to accurately link cell death with behavioral dysfunction or phenotypic variation in vivo . Moreover, chemical treatments, as well as forward and reverse genetic screening, hasten the identification of modifiers that alter neurodegeneration. When combined, these chemical-genetic analyses establish critical threshold states to enhance or reduce cellular stress for dissecting associated pathways. Furthermore, C. elegans can rapidly reveal whether lifespan or healthspan factor into neurodegenerative processes. Here, we outline the methodologies employed to investigate neurodegeneration in C. elegans and highlight numerous studies that exemplify its utility as a pre-clinical intermediary to expedite and inform mammalian translational research.

ABSTRACT Red blood cell distribution width (RDW) is a measurement of the variation in size and volume of red blood cells (RBCs). Increased RDW, indicating a high heterogeneity of RBCs, is prominently associated with a variety of illnesses, especially cardiovascular diseases. However, the significance of this association to the onset and progression of cardiovascular and renal diseases is unknown. We hypothesized that a genetic predisposition for increased RDW is an early risk factor for cardiovascular and renal comorbidities. Since there is no known animal model of increased RDW, we examined a CRISPR/Cas9 gene-edited rat model (Rffl TD ) that presented with features of hematologic abnormalities as well as severe cardiac and renal comorbidities. A mass spectrometry-based quantitative proteomic analysis indicated anemia of these rats, which presented with significant downregulation of hemoglobin and haptoglobin. Decreased hemoglobin and increased RDW were further observed in Rffl TD through complete blood count. Next, a systematic temporal assessment detected an early increased RDW in Rffl TD , which was prior to the development of other comorbidities. The primary mutation of Rffl TD is a 50 bp deletion in a non-coding region, and our study has serendipitously identified this locus as a novel quantitative trait locus (QTL) for RDW. To our knowledge, our study is the first to experimentally pinpoint a QTL for RDW and provides a novel genetic rat model mimicking the clinical association of increased RDW with poor cardio-renal outcome.

ABSTRACT Schizophrenia remains one of the most debilitating human neurodevelopmental disorders, with few effective treatments and striking consequences felt by individuals, communities and society as a whole. As such, there remains a critical need for further investigation into the mechanistic underpinnings of schizophrenia so that novel therapeutic targets can be identified. Because schizophrenia is a highly heritable disorder, genetic risk factors remain an attractive avenue for this research. Given their clear molecular genetic consequences, recurrent microdeletions and duplications, or copy number variants (CNVs), represent one of the most tractable genetic entry points to elucidating these mechanisms. To date, eight CNVs have been shown to significantly increase the risk of schizophrenia. Although rodent models of these CNVs that exhibit behavioral phenotypes have been generated, the underlying molecular mechanisms remain largely elusive. Over the past decades, the zebrafish has emerged as a powerful vertebrate model that has led to fundamental discoveries in developmental neurobiology and behavioral genetics. Here, we review the attributes that make zebrafish exceptionally well suited to investigating individual and combinatorial gene contributions to CNV-mediated brain dysfunction in schizophrenia. With highly conserved genetics and neural substrates, an ever-expanding molecular genetic and imaging toolkit, and ability to perform high-throughput and high-content genetic and pharmacologic screens, zebrafish is poised to generate deep insights into the molecular genetic mechanisms of schizophrenia-associated neurodevelopmental and behavioral deficits, and to facilitate the identification of therapeutic targets.

ABSTRACT Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart ( nsh ), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line–A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.

ABSTRACT Congenital heart defects affect ∼2% of live births and often involve malformations of the semilunar (aortic and pulmonic) valves. We previously reported a highly penetrant GATA4 p.Gly296Ser mutation in familial, congenital atrial septal defects and pulmonic valve stenosis and showed that mice harboring the orthologous G295S disease-causing mutation display not only atrial septal defects, but also semilunar valve stenosis. Here, we aimed to characterize the role of Gata4 in semilunar valve development and stenosis using the Gata4 G295Ski/wt mouse model. GATA4 is highly expressed in developing valve endothelial and interstitial cells. Echocardiographic examination of Gata4 G295Ski/wt mice at 2 months and 1 year of age identified functional semilunar valve stenosis predominantly affecting the aortic valve with distal leaflet thickening and severe extracellular matrix (ECM) disorganization. Examination of the aortic valve at earlier postnatal timepoints demonstrated similar ECM abnormalities consistent with congenital disease. Analysis at embryonic timepoints showed a reduction in aortic valve cushion volume at embryonic day (E)13.5, predominantly affecting the non-coronary cusp (NCC). Although total cusp volume recovered by E15.5, the NCC cusp remained statistically smaller. As endothelial to mesenchymal transition (EMT)-derived cells contribute significantly to the NCC, we performed proximal outflow tract cushion explant assays and found EMT deficits in Gata4 G295Ski/wt embryos along with deficits in cell proliferation. RNA-seq analysis of E15.5 outflow tracts of mutant embryos suggested a disease state and identified changes in genes involved in ECM and cell migration as well as dysregulation of Wnt signaling. By utilizing a mouse model harboring a human disease-causing mutation, we demonstrate a novel role for GATA4 in congenital semilunar valve stenosis. This article has an associated First Person interview with the joint first authors of the paper .

Harold Varmus has made pioneering contributions to our understanding of cancer as a genetic disease. The discovery of the cellular origin of retroviral oncogenes earned him and his long-term collaborator, Michael Bishop, the Lasker Prize for Basic Medical Sciences in 1982 and the Nobel Prize in Physiology and Medicine in 1989. Throughout his career, Varmus has held several leadership roles that shaped science policy in the US and worldwide, and he has been an outspoken advocate for open science. In this interview, he talks (among other things) about the factors that shaped his early career choices, the thrill of scientific discovery, and the importance of including diverse populations in genomic studies of cancer and other diseases.

ABSTRACT The mechanisms underlying diabetes remain unresolved. The Cohen diabetic rat represents a model of diet-induced diabetes, in which the disease is induced after exposure to a diabetogenic diet (DD) in the diabetes-sensitive (CDs/y) but not in the -resistant (CDr/y) strain. Diet imposes a metabolic strain that leads to diabetes in the appropriate genetic background. We previously identified, through whole-genome linkage analysis, a diabetes-related quantitative trait locus on rat chromosome 4 (RNO4), which incorporates NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 ( Ndufa4 ), a nuclear gene that affects mitochondrial function. Here, we sequenced the gene and found a major deletion in CDs/y that leads to lack of expression of the NDUFA4 protein that has been reported to be involved in the activities of mitochondrial complexes I and IV. In the absence of NDUFA4 in the diabetic CDs/y on DD, complex I activity is reduced in comparison to that in nondiabetic CDs/y on regular diet and CDr/y on either diet; complex IV activity is reduced in both strains provided DD, and thus as a result of diet and unrelated to the gene mutation. ATP fails to increase in diabetic CDs/y in response to DD, in comparison to nondiabetic CDr/y on DD. Plasma malondialdehyde levels are elevated in CDs/y on DD, whereas SOD1 and SOD2 levels fail to increase, indicating increased oxidative stress and inability of the pancreas to generate an appropriate antioxidative stress response. These findings suggest that the Ndufa4 mutation in CDs/y on DD is directly associated with mitochondrial dysfunction, which we attribute to the lack of expression of NDUFA4 and to diet, and which prevents the anticipated increase in ATP production. The resulting enhanced oxidative stress impairs the ability of the pancreas to secrete insulin, leading to the development of diabetes. This is the first demonstration of an inherited mutation in a nuclear gene that adversely affects mitochondrial function and promotes diet-induced diabetes.

ABSTRACT Inflammation of the middle ear, known clinically as chronic otitis media, presents in different forms, such as chronic otitis media with effusion (COME; glue ear) and chronic suppurative otitis media (CSOM). These are highly prevalent diseases, especially in childhood, and lead to significant morbidity worldwide. However, much remains unclear about this disease, including its aetiology, initiation and perpetuation, and the relative roles of mucosal and leukocyte biology, pathogens, and Eustachian tube function. Chronic otitis media is commonly modelled in mice but most existing models only partially mimic human disease and many are syndromic. Nevertheless, these models have provided insights into potential disease mechanisms, and have implicated altered immune signalling, mucociliary function and Eustachian tube function as potential predisposing mechanisms. Clinical studies of chronic otitis media have yet to implicate a particular molecular pathway or mechanism, and current human genetic studies are underpowered. We also do not fully understand how existing interventions, such as tympanic membrane repair, work, nor how chronic otitis media spontaneously resolves. This Clinical Puzzle article describes our current knowledge of chronic otitis media and the existing research models for this condition. It also identifies unanswered questions about its pathogenesis and treatment, with the goal of advancing our understanding of this disease to aid the development of novel therapeutic interventions.

ABSTRACT Congenital diaphragmatic hernias (CDHs) and structural anomalies of the diaphragm are a common class of congenital birth defects that are associated with significant morbidity and mortality due to associated pulmonary hypoplasia, pulmonary hypertension and heart failure. In ∼30% of CDH patients, genomic analyses have identified a range of genetic defects, including chromosomal anomalies, copy number variants and sequence variants. The affected genes identified in CDH patients include transcription factors, such as GATA4 , ZFPM2 , NR2F2 and WT1 , and signaling pathway components, including members of the retinoic acid pathway. Mutations in these genes affect diaphragm development and can have pleiotropic effects on pulmonary and cardiac development. New therapies, including fetal endoscopic tracheal occlusion and prenatal transplacental fetal treatments, aim to normalize lung development and pulmonary vascular tone to prevent and treat lung hypoplasia and pulmonary hypertension, respectively. Studies of the association between particular genetic mutations and clinical outcomes should allow us to better understand the origin of this birth defect and to improve our ability to predict and identify patients most likely to benefit from specialized treatment strategies.

ABSTRACT Rheumatoid arthritis is a chronic inflammatory joint disorder characterised by erosive inflammation of the articular cartilage and by destruction of the synovial joints. It is regulated by both genetic and environmental factors, and, currently, there is no preventative treatment or cure for this disease. Genome-wide association studies have identified ∼100 new loci associated with rheumatoid arthritis, in addition to the already known locus within the major histocompatibility complex II region. However, together, these loci account for only a modest fraction of the genetic variance associated with this disease and very little is known about the pathogenic roles of most of the risk loci identified. Here, we discuss how rat models of rheumatoid arthritis are being used to detect quantitative trait loci that regulate different arthritic traits by genetic linkage analysis and to positionally clone the underlying causative genes using congenic strains. By isolating specific loci on a fixed genetic background, congenic strains overcome the challenges of genetic heterogeneity and environmental interactions associated with human studies. Most importantly, congenic strains allow functional experimental studies be performed to investigate the pathological consequences of natural genetic polymorphisms, as illustrated by the discovery of several major disease genes that contribute to arthritis in rats. We discuss how these advances have provided new biological insights into arthritis in humans.

ABSTRACT A variety of inherited animal models with different genetic causes and distinct genetic backgrounds are needed to help dissect the complex genetic etiology of glaucoma. The scarcity of such animal models has hampered progress in glaucoma research. Here, we introduce a new inherited glaucoma model: the inbred mouse strain YBR/EiJ (YBR). YBR mice develop a form of pigmentary glaucoma. They exhibit a progressive age-related pigment-dispersing iris disease characterized by iris stromal atrophy. Subsequently, these mice develop elevated intraocular pressure (IOP) and glaucoma. Genetic mapping studies utilizing YBR as a glaucoma-susceptible strain and C57BL/6J as a glaucoma-resistant strain were performed to identify genetic loci responsible for the iris disease and high IOP. A recessive locus linked to Tyrp1 b on chromosome 4 contributes to iris stromal atrophy and high IOP. However, this is not the only important locus. A recessive locus on YBR chromosome 17 causes high IOP independent of the iris stromal atrophy. In specific eyes with high IOP caused by YBR chromosome 17, the drainage angle (through which ocular fluid leaves the eye) is largely open. The YBR alleles of genes on chromosomes 4 and 17 underlie the development of high IOP and glaucoma but do so through independent mechanisms. Together, these two loci act in an additive manner to increase the susceptibility of YBR mice to the development of high IOP. The chromosome 17 locus is important not only because it causes IOP elevation in mice with largely open drainage angles but also because it exacerbates IOP elevation and glaucoma induced by pigment dispersion. Therefore, YBR mice are a valuable resource for studying the genetic etiology of IOP elevation and glaucoma, as well as for testing new treatments.

ABSTRACT The Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1- null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6 cre produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity.

Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged as a useful model to investigate mycobacterial pathogenesis and treatment. Infection of zebrafish with Mycobacterium marinum , the closest relative of the Mycobacterium tuberculosis complex, recapitulates many aspects of human tuberculosis. The zebrafish model affords optical transparency, abundant genetic tools and in vivo imaging of the progression of infection. Here, we review how the zebrafish– M. marinum system has been deployed to make novel observations about the role of innate immunity, the tuberculous granuloma, and crucial host and bacterial genes. Finally, we assess how these findings relate to human disease and provide a framework for novel strategies to treat tuberculosis.

Connexin40 (Cx40)-containing gap-junction channels are expressed in the atrial myocardium and provide a low-resistance passage for rapid impulse propagation. A germline mutation in the GJA5 gene, which encodes Cx40, resulting in a truncated Cx40 (Q49X) was identified in a large Chinese family with lone (idiopathic) atrial fibrillation (AF). This mutation co-segregated with seven AF probands in an autosomal-dominant way over generations. To test the hypothesis that this Cx40 mutant affects the distribution and function of atrial gap junctions, we studied the Q49X mutant in gap-junction-deficient HeLa and N2A cells. The Q49X mutant, unlike wild-type Cx40, was typically localized in the cytoplasm and failed to form gap-junction plaques at cell-cell interfaces. When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43. Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43. Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum. These findings provide evidence that the Q49X mutant is capable of impairing gap-junction distribution and function of key atrial connexins, which might play a role in the predisposition to and onset of AF.