Large-scale genomic investigation of pediatric cholestasis reveals a novel hepatorenal ciliopathy caused by PSKH1 mutations.

PURPOSE: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly. METHODS: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test. RESULTS: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in 3 consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the 4 families shared a founder homozygous variant, whereas the third and fourth had different homozygous variants in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays. CONCLUSION: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form.

A novel model of nephrotic syndrome results from a point mutation in Lama5 and is modified by genetic background.

Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin ß2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. Here, we have identified a novel missense mutation (E884G) in the uncharacterized L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus mimicking the progression seen in most patients. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background of the mice had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. Thus, our novel model will provide insights into pathologic mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane that may be important in a range of kidney diseases.

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations.

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.

Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing.

Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to 'finding a needle in a haystack'. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.

Modelling age-related metabolic disorders in the mouse.

Ageing can be characterised by a general decline in cellular function, which affects whole-body homoeostasis with metabolic dysfunction-a common hallmark of ageing. The identification and characterisation of the genetic pathways involved are paramount to the understanding of how we age and the development of therapeutic strategies for combating age-related disease. Furthermore, in addition to understanding the ageing process itself, we must understand the interactions ageing has with genetic variation that results in disease phenotypes. The use of model systems such as the mouse, which has a relatively short lifespan, rapid reproduction (resulting in a large number of offspring), well-characterised biology, a fully sequenced genome, and the availability of tools for genetic manipulation is essential for such studies. Here we review the relationship between ageing and metabolism and highlight the need for modelling these processes.

HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

Pleiotropic brain function of whirlin identified by a novel mutation.

Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation (head-bob, hb) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function.

Modeling and Assessing Osteoarthritis in Mice by Destabilization of the Medial Meniscus (DMM).

In this chapter, we describe an induced model of osteoarthritis in mice, frequently employed in the study of this disease. We outline in detail the surgical induction of disease and preparation of samples for histological assessment of disease.

Tissue-specific differences in the assembly of mitochondrial Complex I are revealed by a novel ENU mutation in ECSIT.

AIMS: Mitochondrial Complex I assembly (MCIA) is a multi-step process that necessitates the involvement of a variety of assembly factors and chaperones to ensure that the final active enzyme is correctly assembled. The role of the assembly factor evolutionarily conserved signalling intermediate in the toll (ECSIT) pathway was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesized that many of the known functions of ECSIT were unhindered by the introduction of an ENU-induced mutation, while its role in Complex I assembly was affected on a tissue-specific basis. METHODS AND RESULTS: Here, we describe a mutation in the MCIA factor ECSIT that reveals tissue-specific requirements for ECSIT in Complex I assembly. MCIA is a multi-step process dependent on assembly factors that organize and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU-induced mutation in ECSIT (N209I) that exhibits a profound effect on Complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of Complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, while mitochondria from other tissues were unaffected. CONCLUSIONS: These data suggest that the mechanisms underlying Complex I assembly and activity may have tissue-specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high-energy demands, such as the heart, may utilize assembly factors in different ways to low-energy tissues in order to improve mitochondrial output. These data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying genetic cause.

Chronically elevated branched chain amino acid levels are pro-arrhythmic.

AIMS: Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. METHODS AND RESULTS: We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6-9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4-5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs-leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. CONCLUSIONS: Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.

Modification of an aggressive model of Alport Syndrome reveals early differences in disease pathogenesis due to genetic background.

The link between mutations in collagen genes and the development of Alport Syndrome has been clearly established and a number of animal models, including knock-out mouse lines, have been developed that mirror disease observed in patients. However, it is clear from both patients and animal models that the progression of disease can vary greatly and can be modified genetically. We have identified a point mutation in Col4a4 in mice where disease is modified by strain background, providing further evidence of the genetic modification of disease symptoms. Our results indicate that C57BL/6J is a protective background and postpones end stage renal failure from 7 weeks, as seen on a C3H background, to several months. We have identified early differences in disease progression, including expression of podocyte-specific genes and podocyte morphology. In C57BL/6J mice podocyte effacement is delayed, prolonging normal renal function. The slower disease progression has allowed us to begin dissecting the pathogenesis of murine Alport Syndrome in detail. We find that there is evidence of differential gene expression during disease on the two genetic backgrounds, and that disease diverges by 4 weeks of age. We also show that an inflammatory response with increasing MCP-1 and KIM-1 levels precedes loss of renal function.

Quantifying Microsatellite Mutation Rates from Intestinal Stem Cell Dynamics in Msh2-Deficient Murine Epithelium.

Microsatellite sequences have an enhanced susceptibility to mutation, and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here, we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed, and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus, and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (∼50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2-deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 × 10-2) compared to wild-type epithelium (6.2 × 10-5).

Efficient clearance of opsonised apoptotic cells in the absence of PECAM-1.

It is now accepted that the recognition and uptake of apoptotic cells by phagocytes is a complex process involving a large number of opsonins, receptors and ligands, however the relative contribution of all these molecules are still debated. Here we examined the role of CD31 (PECAM-1) in the recognition/uptake of apoptotic thymocytes by murine bone marrow-derived macrophages (BMDM) in vitro, and by resident peritoneal macrophages in vivo. In the absence of serum, CD31 deficiency, on either the phagocyte or the apoptotic cell, resulted in a reduction in the clearance of apoptotic thymocytes, when a high ratio of apoptotic cells to macrophages was applied. In the presence of serum however there was no discernible contribution of CD31 to the clearance of apoptotic cells by bone marrow-derived macrophages, irrespective of the ratio of cells used. In vivo peritoneal clearance experiments confirmed that in the presence of soluble opsonins CD31 deficiency had no effect on this process. These data suggest that the overall role played by CD31 in the ingestion of apoptotic cells is negligible and most likely overwhelmed by the effects of serum opsonins, such as complement components.

Generation and Identification of Mutations Resulting in Chronic and Age-Related Phenotypes in Mice.

Aging is inevitable, and our society must deal with the consequences: namely, an increased incidence of disease and ill health. Many mouse models of disease are acute or early onset or are induced in young mice, despite the fact that aging is a significant risk factor for a range of significant diseases. To improve modeling of such diseases, we should incorporate aging into our models. Many systems are affected by aging, with a decline in mitochondrial function, an increase in senescence, a loss of resilience, telomere shortening, and a decline in immune function being key factors in the increased susceptibility to disease that is associated with aging. To develop novel models of age-related disease, we undertook a phenotype-driven screen of a pipeline of mutagenized mice. Here, we describe some of the underlying protocols and outline important aspects to consider when studying aged mice. © 2018 by John Wiley & Sons, Inc.

Studying Osteoarthritis Pathogenesis in Mice.

With the increasing availability and complexity of mouse models of disease, either spontaneous or induced, there is a concomitant increase in their use in the analysis of pathogenesis. Among such diseases is osteoarthritis, a debilitating disease with few treatment options. While advances in our understanding of the pathogenesis of osteoarthritis has advanced through clinical investigations and genome-wide association studies, there is still a large gap in our knowledge, hindering advances in therapy. Patient samples are available ex vivo, but these are generally in the very late stages of disease. However, with mice, we are able to induce disease at a defined time and track the progression in vivo and ex vivo, from inception to end stage, to delineate the processes involved in disease development. © 2018 by John Wiley & Sons, Inc.

Mouse Idh3a mutations cause retinal degeneration and reduced mitochondrial function.

Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one ß and one γ subunit. Loss-of-function and missense mutations in both IDH3A and IDH3B have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Idh3a Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months, but no other tissues appear to be affected. We produced a knockout of Idh3a and found that homozygous mice do not survive past early embryogenesis. Idh3a-/E229K compound heterozygous mutants exhibit a more severe retinal degeneration compared with Idh3aE229K/E229K homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both Idh3aE229K/E229K and Idh3a-/E229K cells. Loss-of-function Idh3b mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in Idh3a mutant mice.This article has an associated First Person interview with the first author of the paper.

An N-Ethyl-N-Nitrosourea (ENU) Mutagenized Mouse Model for Autosomal Dominant Nonsyndromic Kyphoscoliosis Due to Vertebral Fusion.

Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as Hvf. Micro-computed tomography (µCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. µCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized Hvf to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

With mouse age comes wisdom: A review and suggestions of relevant mouse models for age-related conditions.

Ageing is a complex multifactorial process that results in many changes in physiological changes processes that ultimately increase susceptibility to a wide range of diseases. As such an ageing population is resulting in a pressing need for more and improved treatments across an assortment of diseases. Such treatments can come from a better understanding of the pathogenic pathways which, in turn, can be derived from models of disease. Therefore the more closely the model resembles the disease situation the more likely relevant the data will be that is generated from them. Here we review the state of knowledge of mouse models of a range of diseases and aspects of an ageing physiology that are all germane to ageing. We also give recommendations on the most common mouse models on their relevance to the clinical situations occurring in aged patients and look forward as to how research in ageing models can be carried out. As we continue to elucidate the pathophysiology of disease, often through mouse models, we also learn what is needed to refine these models. Such factors can include better models, reflecting the ageing patient population, or a better phenotypic understanding of existing models.

Genome-wide association of multiple complex traits in outbred mice by ultra-low-coverage sequencing.

Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution and the need for population-specific genotyping arrays and haplotype reference panels. Here we combine low-coverage (0.15×) sequencing with a new method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at a 5% false discovery rate. Gene-level mapping resolution was achieved at about one-fifth of the loci, implicating Unc13c and Pgc1a at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how genome-wide association studies can be extended via low-coverage sequencing to species with highly recombinant outbred populations.