Human Disease Phenotypes Research Articles

Professor Jean Aicardi

Professor Jean Aicardi

Tailored Pig Models for Preclinical Efficacy and Safety Testing of Targeted Therapies.

Despite enormous advances in translational biomedical research, there remains a growing demand for improved animal models of human disease. This is particularly true for diseases where rodent models do not reflect the human disease phenotype. Compared to rodents, pig anatomy and physiology are more similar to humans in cardiovascular, immune, respiratory, skeletal muscle, and metabolic systems. Importantly, efficient and precise techniques for genetic engineering of pigs are now available, facilitating the creation of tailored large animal models that mimic human disease mechanisms at the molecular level. In this article, the benefits of genetically engineered pigs for basic and translational research are exemplified by a novel pig model of Duchenne muscular dystrophy and by porcine models of cystic fibrosis. Particular emphasis is given to potential advantages of using these models for efficacy and safety testing of targeted therapies, such as exon skipping and gene editing, for example, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system. In general, genetically tailored pig models have the potential to bridge the gap between proof-of-concept studies in rodents and clinical trials in patients, thus supporting translational medicine.

MicroRNA (miRNA)-Mediated Pathogenetic Signaling in Alzheimer’s Disease (AD)

Alzheimer's disease (AD) is an expanding health and socioeconomic concern in industrialized societies, and the leading cause of intellectual impairment in our aging population. The cause of AD remains unknown, and there are currently no effective treatments to stop or reverse the progression of this uniquely human and age-related neurological disorder. Elucidation of the AD mechanism and factors that contribute to the initiation, progression, and spreading of this chronic and fatal neurodegeneration will ultimately result in improved and effective diagnostics and therapeutic strategies.microRNAs (miRNAs) comprise a relatively recently discovered category of 20-24 nucleotide non-coding RNAs that function post-transcriptionally in shaping the transcriptome of the cell, and in doing so, contribute to the molecular-genetics and phenotype of human CNS health and disease. To date about 2550 unique mature human miRNAs have been characterized, however only highly selected miRNA populations appear to be enriched in different anatomical compartments within the CNS.This general commentary for the 'Special Issue: 40th Year of Neurochemical Research' will bring into perspective (i) some very recent findings on the extraordinary biophysics and signaling properties of CNS miRNA in AD and aging human brain; (ii) how specific intrinsic biophysical attributes of miRNAs may play defining roles in the establishment, proliferation and spreading of the AD phenotype; and (iii) how miRNAs can serve as prospective therapeutic targets and biomarkers potentially useful in the clinical management of this terminal neurological disease whose incidence in our rapidly aging population is reaching epidemic proportions.

Mouse Models of Stargardt 3 Dominant Macular Degeneration.

Stargardt type 3 macular degeneration is dependent on a dominant defect in a single gene, ELOVL4 (elongase of very long chain fatty acids 4). The encoded enzyme, ELOVL4, is required for the synthesis of very long chain polyunsaturated fatty acids (VLC-PUFAs), a rare class of >C24 lipids. In vitro expression studies suggest that mutated ELOVL4(STGD3) proteins fold improperly, resulting in ER stress and formation of cytosolic aggresomes of wild type and mutant ELOVL4. Although a number of mouse models have been developed to determine whether photoreceptor cell loss in STGD3 results from depletion of VLC-PUFAs, aggresome-dependent cell stress or a combination of these two factors, none of these models adequately recapitulates the disease phenotype in humans. Thus, the precise molecular mechanism by which ELOVL4 mutation causes photoreceptor degeneration in mice and in human patients remains to be characterized. This mini review compares and evaluates current STGD3 mouse models and determines what conclusions can be drawn from past work.

Metalloprotease OMA1 Fine-tunes Mitochondrial Bioenergetic Function and Respiratory Supercomplex Stability.

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism – intramitochondrial quality control (IMQC) – is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.

Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals.

The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.

Genetic Control of Chromatin States in Humans Involves Local and Distal Chromosomal Interactions

Deciphering the impact of genetic variants on gene regulation is fundamental to understanding human disease. Although gene regulation often involves long-range interactions, it is unknown to what extent non-coding genetic variants influence distal molecular phenotypes. Here, we integrate chromatin profiling for three histone marks in lymphoblastoid cell lines (LCLs) from 75 sequenced individuals with LCL-specific Hi-C and ChIA-PET-based chromatin contact maps to uncover one of the largest collections of local and distal histone quantitative trait loci (hQTLs). Distal QTLs are enriched within topologically associated domains and exhibit largely concordant variation ofchromatin state coordinated by proximal and distalnon-coding genetic variants. Histone QTLs are enriched for common variants associated with autoimmune diseases and enable identification of putative target genes of disease-associated variants from genome-wide association studies. These analyses provide insights into how genetic variation can affect human disease phenotypes by coordinated changes in chromatin at interacting regulatory elements.

The clinical relevance of animal models in Sjögren’s syndrome: the interferon signature from mouse to man

Mouse models have been widely used to elucidate the pathogenic mechanisms of human diseases. The advantages of using these models include the ability to study different stages of the disease with particular respect to specific target organs, to focus on the role of specific pathogenic factors and to investigate the effect of possible therapeutic interventions. Sjögren’s syndrome (SS) is a systemic autoimmune disease, characterised by lymphocytic infiltrates in the salivary and lacrimal glands. To date, effective therapy is not available and treatment has been mainly symptomatic. Ongoing studies in murine models are aimed at developing more effective and targeted therapies in SS. The heterogeneity of SS will most probably benefit from optimising therapies, tailored to specific subgroups of the disease. In this review, we provide our perspective on the importance of subdividing SS patients according to their interferon signature, and recommend choosing appropriate mouse models for interferon-positive and interferon-negative SS subtypes. Murine models better resembling human-disease phenotypes will be essential in this endeavour.

Phenome-driven disease genetics prediction toward drug discovery.

Motivation: Discerning genetic contributions to diseases not only enhances our understanding of disease mechanisms, but also leads to translational opportunities for drug discovery. Recent computational approaches incorporate disease phenotypic similarities to improve the prediction power of disease gene discovery. However, most current studies used only one data source of human disease phenotype. We present an innovative and generic strategy for combining multiple different data sources of human disease phenotype and predicting disease-associated genes from integrated phenotypic and genomic data.Results: To demonstrate our approach, we explored a new phenotype database from biomedical ontologies and constructed Disease Manifestation Network (DMN). We combined DMN with mimMiner, which was a widely used phenotype database in disease gene prediction studies. Our approach achieved significantly improved performance over a baseline method, which used only one phenotype data source. In the leave-one-out cross-validation and de novo gene prediction analysis, our approach achieved the area under the curves of 90.7% and 90.3%, which are significantly higher than 84.2% (P < e−4) and 81.3% (P < e−12) for the baseline approach. We further demonstrated that our predicted genes have the translational potential in drug discovery. We used Crohn’s disease as an example and ranked the candidate drugs based on the rank of drug targets. Our gene prediction approach prioritized druggable genes that are likely to be associated with Crohn’s disease pathogenesis, and our rank of candidate drugs successfully prioritized the Food and Drug Administration-approved drugs for Crohn’s disease. We also found literature evidence to support a number of drugs among the top 200 candidates. In summary, we demonstrated that a novel strategy combining unique disease phenotype data with system approaches can lead to rapid drug discovery.Availability and implementation: nlp.case.edu/public/data/DMNContact: rxx@case.edu

Multiple Changes of Gene Expression and Function Reveal Genomic and Phenotypic Complexity in SLE-like Disease.

The complexity of clinical manifestations commonly observed in autoimmune disorders poses a major challenge to genetic studies of such diseases. Systemic lupus erythematosus (SLE) affects humans as well as other mammals, and is characterized by the presence of antinuclear antibodies (ANA) in patients’ sera and multiple disparate clinical features. Here we present evidence that particular sub-phenotypes of canine SLE-related disease, based on homogenous (ANAH) and speckled ANA (ANAS) staining pattern, and also steroid-responsive meningitis-arteritis (SRMA) are associated with different but overlapping sets of genes. In addition to association to certain MHC alleles and haplotypes, we identified 11 genes (WFDC3, HOMER2, VRK1, PTPN3, WHAMM, BANK1, AP3B2, DAPP1, LAMTOR3, DDIT4L and PPP3CA) located on five chromosomes that contain multiple risk haplotypes correlated with gene expression and disease sub-phenotypes in an intricate manner. Intriguingly, the association of BANK1 with both human and canine SLE appears to lead to similar changes in gene expression levels in both species. Our results suggest that molecular definition may help unravel the mechanisms of different clinical features common between and specific to various autoimmune disease phenotypes in dogs and humans.

Erythrokeratodermia Variabilis et Progressiva Allelic to Oculo-Dento-Digital Dysplasia

Erythrokeratodermia variabilis et progressiva (EKVP) is a genodermatosis with clinical and genetic heterogeneity, most often transmitted in an autosomal dominant manner, caused by mutations in GJB3 and GJB4 genes encoding connexins (Cx)31 and 30.3, respectively. In this issue, Boyden et al. (2015) report for the first time de novo dominant mutations in GJA1 encoding the ubiquitous Cx43 in patients with EKVP. These results expand the genetic heterogeneity of EKVP and the human disease phenotypes associated with GJA1 mutations. They disclose that EKVP is allelic to oculo-dento-digital dysplasia, a rare syndrome previously known to be caused by dominant GJA1 mutations.

RCARE: RNA Sequence Comparison and Annotation for RNA Editing.

The post-transcriptional sequence modification of transcripts through RNA editing is an important mechanism for regulating protein function and is associated with human disease phenotypes. The identification of RNA editing or RNA-DNA difference (RDD) sites is a fundamental step in the study of RNA editing. However, a substantial number of false-positive RDD sites have been identified recently. A major challenge in identifying RDD sites is to distinguish between the true RNA editing sites and the false positives. Furthermore, determining the location of condition-specific RDD sites and elucidating their functional roles will help toward understanding various biological phenomena that are mediated by RNA editing. The present study developed RNA-sequence comparison and annotation for RNA editing (RCARE) for searching, annotating, and visualizing RDD sites using thousands of previously known editing sites, which can be used for comparative analyses between multiple samples. RCARE also provides evidence for improving the reliability of identified RDD sites. RCARE is a web-based comparison, annotation, and visualization tool, which provides rich biological annotations and useful summary plots. The developers of previous tools that identify or annotate RNA-editing sites seldom mention the reliability of their respective tools. In order to address the issue, RCARE utilizes a number of scientific publications and databases to find specific documentations respective to a particular RNA-editing site, which generates evidence levels to convey the reliability of RCARE. Sequence-based alignment files can be converted into VCF files using a Python script and uploaded to the RCARE server for further analysis. RCARE is available for free at http://www.snubi.org/software/rcare/.

Network-based Phenome-Genome Association Prediction by Bi-Random Walk.

MotivationThe availability of ontologies and systematic documentations of phenotypes and their genetic associations has enabled large-scale network-based global analyses of the association between the complete collection of phenotypes (phenome) and genes. To provide a fundamental understanding of how the network information is relevant to phenotype-gene associations, we analyze the circular bigraphs (CBGs) in OMIM human disease phenotype-gene association network and MGI mouse phentoype-gene association network, and introduce a bi-random walk (BiRW) algorithm to capture the CBG patterns in the networks for unveiling human and mouse phenome-genome association. BiRW performs separate random walk simultaneously on gene interaction network and phenotype similarity network to explore gene paths and phenotype paths in CBGs of different sizes to summarize their associations as predictions.ResultsThe analysis of both OMIM and MGI associations revealed that majority of the phenotype-gene associations are covered by CBG patterns of small path lengths, and there is a clear correlation between the CBG coverage and the predictability of the phenotype-gene associations. In the experiments on recovering known associations in cross-validations on human disease phenotypes and mouse phenotypes, BiRW effectively improved prediction performance over the compared methods. The constructed global human disease phenome-genome association map also revealed interesting new predictions and phenotype-gene modules by disease classes.

Initial Characterization of A Mouse Model of Severe Asthma

Severe asthma patients have symptoms that are frequently hard to manage with low dose inhaled corticosteroids (ICS) and often need additional high dose oral steroids. Neutrophilic airway inflammation is also associated with corticosteroid insensitivity in this population. We have investigated the effect of inhaled and oral steroids, a p38 MAPK inhibitor and an anti-mouse IL-17 monoclonal antibody (mAb) in a mouse severe asthma model. Methods BALB/c mice were sensitised with house dust mite (HDM, 100µg s.c.) emulsified in Complete Freunds Adjuvant (CFA) and 14 days later challenged with 100µg HDM intra nasally. Mice were dosed with steroid (Budesonide 0.1, 0.3, 1mg/kg i.n. or Dexamethasone 0.3, 1, 3mg/kg p.o.), or the p38 MAPK inhibitor BIRB-796 (3mg/kg, p.o.), or anti-IL-17 mAb (200µg/mouse, s.c.), or vehicle prior to and/or following HDM-challenge. The lungs were lavaged and differential cell numbers determined. Results HDM-challenge caused a significant increase in both neutrophils and eosinophils in the lung lavage. All Budesonide doses failed to inhibit the neutrophilia and only the maximum dose (1mg/kg) inhibited eosinophilia (p<0.05, 47% inhibition). Only the highest dose of Dexamethasone (3mg/kg) inhibited both the neutrophilia (p<0.0001, 81% inhibition) and eosinophilia (p<0.0001, 85% inhibition). BIRB-796 did not inhibit either the neutrophilia and eosinophilia. However, the anti-IL-17 mAb inhibited the neutrophilia (p<0.0001, 46% inhibition), but not the eosinophilia. In summary, the CFA/HDM severe asthma model is insensitive to ICS but sensitive to high doses of oral steroid, thus mimicking phenotypes of human disease. In addition, the neutrophilic inflammation is inhibited with anti-IL-17 mAb treatment. The model may be useful to investigate potential mechanisms. Funded by Argenta

SubmiRine: assessing variants in microRNA targets using clinical genomic data sets.

MicroRNAs (miRNAs) regulate gene expression by binding to partially complementary sequences on target mRNA transcripts, thereby causing their degradation, deadenylation, or inhibiting their translation. Genomic variants can alter miRNA regulation by modifying miRNA target sites, and multiple human disease phenotypes have been linked to such miRNA target site variants (miR-TSVs). However, systematic genome-wide identification of functional miR-TSVs is difficult due to high false positive rates; functional miRNA recognition sequences can be as short as six nucleotides, with the human genome encoding thousands of miRNAs. Furthermore, while large-scale clinical genomic data sets are becoming increasingly commonplace, existing miR-TSV prediction methods are not designed to analyze these data. Here, we present an open-source tool called SubmiRine that is designed to perform efficient miR-TSV prediction systematically on variants identified in novel clinical genomic data sets. Most importantly, SubmiRine allows for the prioritization of predicted miR-TSVs according to their relative probability of being functional. We present the results of SubmiRine using integrated clinical genomic data from a large-scale cohort study on chronic obstructive pulmonary disease (COPD), making a number of high-scoring, novel miR-TSV predictions. We also demonstrate SubmiRine's ability to predict and prioritize known miR-TSVs that have undergone experimental validation in previous studies.

PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia.

Non-progressive cerebellar ataxias are a rare group of disorders that comprise approximately 10% of static infantile encephalopathies. We report the identification of mutations in PMPCA in 17 patients from four families affected with cerebellar ataxia, including the large Lebanese family previously described with autosomal recessive cerebellar ataxia and short stature of Norman type and localized to chromosome 9q34 (OMIM #213200). All patients present with non-progressive cerebellar ataxia, and the majority have intellectual disability of variable severity. PMPCA encodes α-MPP, the alpha subunit of mitochondrial processing peptidase, the primary enzyme responsible for the maturation of the vast majority of nuclear-encoded mitochondrial proteins, which is necessary for life at the cellular level. Analysis of lymphoblastoid cells and fibroblasts from patients homozygous for the PMPCA p.Ala377Thr mutation and carriers demonstrate that the mutation impacts both the level of the alpha subunit encoded by PMPCA and the function of mitochondrial processing peptidase. In particular, this mutation impacts the maturation process of frataxin, the protein which is depleted in Friedreich ataxia. This study represents the first time that defects in PMPCA and mitochondrial processing peptidase have been described in association with a disease phenotype in humans.

Translating genetic findings in hereditary nephrotic syndrome: the missing loops.

Nephrotic syndrome (NS) is a clinicopathological entity characterized by proteinuria, hypoalbuminemia, peripheral edema, and hyperlipidemia. It is the most common cause of glomerular disease in children and adults. Although the molecular pathogenesis of NS is not completely understood, data from the study of familial NS suggest that it is a "podocytopathy." Virtually all of the genes mutated in hereditary NS localize to the podocyte or its secreted products and the slit diaphragm. Since the completion of human genome sequence and the advent of next generation sequencing, at least 29 causes of single-gene NS have been identified. However, these findings have not been matched by therapeutic advances owing to suboptimal in vitro and in vivo models for the study of human glomerular disease and podocyte injury phenotypes. Multidisciplinary collaboration between clinicians, geneticists, cell biologists, and molecular physiologists has the potential to overcome this barrier and thereby speed up the translation of genetic findings into improved patient care.

Human disease phenotypes associated with mutations in TREX1.

Considering that it is a single exon gene encoding a 314 amino acid protein, the genotype-phenotype landscape of TREX1 is remarkably complex. Here we briefly describe the human diseases so-far associated with mutations in TREX1, which include Aicardi-Goutières syndrome, familial chilblain lupus, systemic lupus erythematosus and retinal vasculopathy with cerebral leukodystrophy.

CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes

The era of genomics has demanded the development of more efficient and timesaving approaches to validate gene function in disease. Here, we utilized the CRISPR-Cas9 system to generate Kcnj13 mutant mice by zygote injection to verify the pathogenic role of human KCNJ13, mutations of which are thought to cause Leber congenital amaurosis (LCA), an early-onset form of blindness. We found that complete loss of Kcnj13 is likely postnatal lethal. Among surviving F0-generation mice examined, 80% show mosaic KCNJ13 expression in the retinal pigment epithelium (RPE). Mosaic expression correlates with decreased response to light and photoreceptor degeneration, indicating that Kcnj13 mutant mice mimic human KCNJ13-related LCA disease. Importantly, mosaic animals enable us to directly compare Kcnj13 mutant and wild-type RPE cells in the same eye. We found that RPE cells lacking KCNJ13 protein still survive but overlying photoreceptors exhibit cell degeneration. At the same time, wild-type RPE cells can rescue neighboring photoreceptor cells that overlie mutant RPE cells. These results suggest that KCNJ13 expression is required for RPE cells to maintain photoreceptor survival. Moreover, we show that CRISPR-Cas9 engineered mosaicism can be used to rapidly test candidate gene function in vivo.

HPOSim: an R package for phenotypic similarity measure and enrichment analysis based on the human phenotype ontology.

BackgroundPhenotypic features associated with genes and diseases play an important role in disease-related studies and most of the available methods focus solely on the Online Mendelian Inheritance in Man (OMIM) database without considering the controlled vocabulary. The Human Phenotype Ontology (HPO) provides a standardized and controlled vocabulary covering phenotypic abnormalities in human diseases, and becomes a comprehensive resource for computational analysis of human disease phenotypes. Most of the existing HPO-based software tools cannot be used offline and provide only few similarity measures. Therefore, there is a critical need for developing a comprehensive and offline software for phenotypic features similarity based on HPO.ResultsHPOSim is an R package for analyzing phenotypic similarity for genes and diseases based on HPO data. Seven commonly used semantic similarity measures are implemented in HPOSim. Enrichment analysis of gene sets and disease sets are also implemented, including hypergeometric enrichment analysis and network ontology analysis (NOA).ConclusionsHPOSim can be used to predict disease genes and explore disease-related function of gene modules. HPOSim is open source and freely available at SourceForge (https://sourceforge.net/p/hposim/).