Human Disease Phenotypes Research Articles

Translatable pathways classification (TransPath-C) for inferring processes germane to human biology from animal studies data: example application in neurobiology.

How to translate insights gained from studies in one organismal species for what is most likely to be germane in another species, such as from mice to humans, is a ubiquitous challenge in basic biology as well as biomedicine. This is an especially difficult problem when there are few molecular features that are obviously important in both species for a given phenotype of interest. Neuropathologies are a prominent realm of this complication. Schizophrenia is complex psychiatric disorder that affects 1% of the population. Many genetic factors have been proposed to drive the development of schizophrenia, and the 22q11 microdeletion (MD) syndrome has been shown to dramatically increase this risk. Due to heterogeneity of presentation of symptoms, diagnosis and formulation of treatment options for patients can often be delayed, and there is an urgent need for novel therapeutics directed toward the treatment of schizophrenia. Here, we present a novel computational approach, Translational Pathways Classification (TransPath-C), that can be used to identify shared pathway dysregulation between mouse models and human schizophrenia cohorts. This method uses variation of pathway activation in the mouse model to predict both mouse and human disease phenotype. Analysis of shared dysregulated pathways called out by both the mouse and human classifiers of TransPath-C can identify pathways that can be targeted in both preclinical and human cohorts of schizophrenia. In application to the 22q11 MD mouse model, our findings suggest that PAR1 pathway activation found upregulated in this mouse phenotype is germane for the corresponding human schizophrenia cohort such that inhibition of PAR1 may offer a novel therapeutic target.

A Novel SCA3 Knock-in Mouse Model Mimics the Human SCA3 Disease Phenotype Including Neuropathological, Behavioral, and Transcriptional Abnormalities Especially in Oligodendrocytes

Spinocerebellar ataxia type 3 is the most common autosomal dominant inherited ataxia worldwide, caused by a CAG repeat expansion in the Ataxin-3 gene resulting in a polyglutamine (polyQ)-expansion in the corresponding protein. The disease is characterized by neuropathological, phenotypical, and specific transcriptional changes in affected brain regions. So far, there is no mouse model available representing all the different aspects of the disease, yet highly needed for a better understanding of the disease pathomechanisms. Here, we characterized a novel Ataxin-3 knock-in mouse model, expressing a heterozygous or homozygous expansion of 304 CAACAGs in the murine Ataxin-3 locus using biochemical, behavioral, and transcriptomic approaches. We compared neuropathological, and behavioral features of the new knock-in model with the in SCA3 research mostly used YAC84Q mouse model. Further, we compared transcriptional changes found in cerebellar samples of the SCA3 knock-in mice and post-mortem human SCA3 patients. The novel knock-in mouse is characterized by the expression of a polyQ-expansion in the murine Ataxin-3 protein, leading to aggregate formation, especially in brain regions known to be vulnerable in SCA3 patients, and impairment of Purkinje cells. Along these neuropathological changes, the mice showed a reduction in body weight accompanied by gait and balance instability. Transcriptomic analysis of cerebellar tissue revealed age-dependent differential expression, enriched for genes attributed to myelinating oligodendrocytes. Comparing these changes with those found in cerebellar tissue of SCA3 patients, we discovered an overlap of differentially expressed genes pointing towards similar gene expression perturbances in several genes linked to myelin sheaths and myelinating oligodendrocytes.

Generation and phenotyping of a novel knock-in mouse model of desmoplakin dependent arrhythmogenic cardiomyopathy

Abstract Background Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disorder, mainly caused by mutations in genes encoding desmosomal proteins, and accounts for most stress-related arrhythmic sudden cardiac deaths (SCD) in the young and athletes. The AC myocardium is hallmarked by cardiomyocyte (CM) death and fibro-fatty replacement, which generate a pro-arrhythmogenic substrate. Several pathogenetic factors in AC remain obscure and better understanding of the disease mechanisms is required to develop novel efficacious therapies to prevent SCD, which are sorely missing. The lexical analogy between desmosomes and desmosomal proteins has originally biased AC research towards CMs, the paradigmatic desmosome-bearing cells in heart. However, the myocardium is composed by different cell types, many of which express desmosomal proteins, albeit in the absence of desmosomes, including CMs, sympathetic neurons, vascular cells and fibroblasts. Notably, AC mutations are transmitted at germline, and thus may manifest in all cell types expressing desmosomal proteins. This might explain why the majority of preclinical AC models, using CM specific over-expression or deletion of the disease-causing mutation, failed to fully recapitulate the human disease phenotype. Hypothesis On these bases, we aimed to generate a knock-in (KI) AC mouse model for comprehensively studying AC pathogenesis. Methods As Desmoplakin (DSP) mutations occur in a large part of the Italian AC population, we used CRISP/Cas9 to generate a KI mouse strain harboring the Serine-to-Alanine substitution of S311, the murine homolog of human S299 [Bauce et al, 2005]. We successfully obtained DSPS311A/WT KI founders, which were viable and fertile and after backcrossing for >10 generations, used to expand the new mouse strain. Mouse cardiac phenotype was characterized, at different stages (1,2,4,6,9 mo.) by functional (i.e. ECHO, telemetry-ECG, chronic exercise) and structural (i.e. EM, standard histology, confocal IF, TUNEL assay) analyses. Molecular/biochemical analyses probed the state of the main pathways involved in AC. Results Our analyses showed that, starting from 4 mo., DSP homozygous KI mice display contractile dysfunction, worsening during aging, and fibrotic myocardial remodelling with focal fatty lesions, accompanied by frequent arrhythmic beats, which become sustained ventricular arrhythmias upon Noradrenaline administration. Hearts showed desmosome alterations, particularly at advanced disease stages, and lateralization of cx43, which corresponded to the phenotype of human AC hearts. Heterozygous mice showed similar alterations, which only took longer to appear. Exercise accelerated disease progression and increased the incidence of SCD (DSPS311A: SCD=63%, n=11; ctrls: SCD=8%, n=12). Conclusion Our KI mice replicate the clinical and pathological phenotype of DSP-linked biventricular AC and are thus suited for the mechanistic study of the multicellular origin of the disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PRIN Miur 2015

Metabolic treatment of an electrical disease? Beneficial APD/QT prolongation by L-Carnitine in transgenic SQT1 rabbit models

Abstract Background Short-QT syndrome 1 (SQT1) is a genetic cardiac channelopathy caused by gain-of-function mutations (KCNH2-N588K) in HERG/IKr, that leads to shortened QT-interval, increased risk for arrhythmias and sudden cardiac death (SCD). An acquired form of SQTS has been described in patients with primary (genetic) carnitine-deficiency, indicating that carnitine might affect cardiac repolarization. Purpose We aimed to investigate potential beneficial (APD/QT-prolonging) effect of L-Carnitine in (genetic) SQTS using transgenic SQT1 rabbits that mimic the human disease phenotype. Methods Effects of L-carnitine on cardiac repolarisation were assessed in adult wildtype (WT) and transgenic SQT1 rabbits (KCNH2-N588K) using in vivo ECG and ex vivo Langendorff-perfused whole-heart or isolated ventricular cardiomyocyte action potential (AP) recordings. Effects on ion currents were assessed by whole-cell patch-clamping. Results In vivo, the heart-rate corrected QT index (QTi) was prolonged significantly by L-carnitine both in WT (QTi, baseline 102.7%±4.9 vs. L-carnitine 106.9%±6.2, p<0.05, n=12) and SQT1 (QTi, baseline 94.8%±7.4 vs. L-carnitine 99.5%±8.2, p<0.05, n=13), leading to normalisation of QTi in SQT1. Ex vivo, whole-heart monophasic and cellular APs were also significantly prolonged by L-carnitine in WT and SQT1 (change in monophasic APD75, ms, WT +13.9±4.4, SQT1 +9.9±7.0; change in cellular APD90, %, WT +10.4%, SQT1 +10.4%, all p<0.05). As underlying mechanisms, we identified acute effects on the main repolarizing ion currents IKr and IKs: IKr-steady, which is significantly increased in SQT1 contributing to accelerated repolarization, was reduced by L-carnitine (WT: −23%, SQT1: −16%). Moreover, L-carnitine accelerated the deactivation kinetics of IKr - while no change was observed in IKr-tail. In addition, IKs-steady was decreased by L-carnitine in SQT1 and WT cardiomyocytes. Conclusion L-carnitine prolongs/normalizes QT and APD in transgenic SQT1 rabbits by decreasing the pathologically increased IKr-steady and also IKs-steady and may therefore serve as potential future anti-arrhythmic therapy in SQTS. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.

PurposeThe Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice.MethodsImmortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ.ResultsEven though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss.ConclusionsMitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.

Circadian PERformance in breast cancer: a germline and somatic genetic study of PER3VNTR polymorphisms and gene co-expression

Polymorphisms in the PER3 gene have been associated with several human disease phenotypes, including sleep disorders and cancer. In particular, the long allele of a variable number of tandem repeat (VNTR) polymorphism has been previously linked to an increased risk of breast cancer. Here we carried out a combined germline and somatic genetic analysis of the role of the PER3VNRT polymorphism in breast cancer. The combined data from 8284 individuals showed a non-significant trend towards increased breast cancer risk in the 5-repeat allele homozygous carriers (OR = 1.17, 95% CI: 0.97–1.42). We observed allelic imbalance at the PER3 locus in matched blood and tumor DNA samples, showing a significant retention of the long variant (risk) allele in tumor samples, and a preferential loss of the short repetition allele (p = 0.0005). Gene co-expression analysis in healthy and tumoral breast tissue samples uncovered significant associations between PER3 expression levels with those from genes which belong to several cancer-associated pathways. Finally, relapse-free survival (RFS) analysis showed that low expression levels of PER3 were linked to a significant lower RSF in luminal A (p = 3 × 10−12) but not in the rest of breast cancer subtypes.

TREX1 as a Novel Immunotherapeutic Target.

Mutations in the TREX1 3’ → 5’ exonuclease are associated with a spectrum of autoimmune disease phenotypes in humans and mice. Failure to degrade DNA activates the cGAS-STING DNA-sensing pathway signaling a type-I interferon (IFN) response that ultimately drives immune system activation. TREX1 and the cGAS-STING DNA-sensing pathway have also been implicated in the tumor microenvironment, where TREX1 is proposed to degrade tumor-derived DNA that would otherwise activate cGAS-STING. If tumor-derived DNA were not degraded, the cGAS-STING pathway would be activated to promote IFN-dependent antitumor immunity. Thus, we hypothesize TREX1 exonuclease inhibition as a novel immunotherapeutic strategy. We present data demonstrating antitumor immunity in the TREX1 D18N mouse model and discuss theory surrounding the best strategy for TREX1 inhibition. Potential complications of TREX1 inhibition as a therapeutic strategy are also discussed.

Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator.

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.

The Role of the U5 snRNP in Genetic Disorders and Cancer.

Pre-mRNA splicing is performed by the spliceosome, a dynamic macromolecular complex consisting of five small uridine-rich ribonucleoprotein complexes (the U1, U2, U4, U5, and U6 snRNPs) and numerous auxiliary splicing factors. A plethora of human disorders are caused by genetic variants affecting the function and/or expression of splicing factors, including the core snRNP proteins. Variants in the genes encoding proteins of the U5 snRNP cause two distinct and tissue-specific human disease phenotypes – variants in PRPF6, PRPF8, and SNRP200 are associated with retinitis pigmentosa (RP), while variants in EFTUD2 and TXNL4A cause the craniofacial disorders mandibulofacial dysostosis Guion-Almeida type (MFDGA) and Burn-McKeown syndrome (BMKS), respectively. Furthermore, recurrent somatic mutations or changes in the expression levels of a number of U5 snRNP proteins (PRPF6, PRPF8, EFTUD2, DDX23, and SNRNP40) have been associated with human cancers. How and why variants in ubiquitously expressed spliceosome proteins required for pre-mRNA splicing in all human cells result in tissue-restricted disease phenotypes is not clear. Additionally, why variants in different, yet interacting, proteins making up the same core spliceosome snRNP result in completely distinct disease outcomes – RP, craniofacial defects or cancer – is unclear. In this review, we define the roles of different U5 snRNP proteins in RP, craniofacial disorders and cancer, including how disease-associated genetic variants affect pre-mRNA splicing and the proposed disease mechanisms. We then propose potential hypotheses for how U5 snRNP variants cause tissue specificity resulting in the restricted and distinct human disorders.

Targeted Gene Editing in Porcine Spermatogonia.

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.

ABSTRACTRetinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.

The Population-Specific Impact of Neandertal Introgression on Human Disease.

Since the discovery of admixture between modern humans and Neandertals, multiple studies investigated the effect of Neandertal-derived DNA on human disease and nondisease phenotypes. These studies have linked Neandertal ancestry to skin- and hair-related phenotypes, immunity, neurological, and behavioral traits. However, these inferences have so far been limited to cohorts with participants of European ancestry. Here, I analyze summary statistics from 40 disease GWAS (genome-wide association study) cohorts of ∼212,000 individuals provided by the Biobank Japan Project for phenotypic effects of Neandertal DNA. I show that Neandertal DNA is associated with autoimmune diseases, prostate cancer and type 2 diabetes. Many of these disease associations are linked to population-specific Neandertal DNA, highlighting the importance of studying a wider range of ancestries to characterize the phenotypic legacy of Neandertals in people today.

CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia

β-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent β-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, β-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit β-globin gene HBB2 with high efficiency and generated a β-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of β-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a β-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of β-thalassemia and associated complications.

Direct Neuronal Reprogramming of Common Marmoset Fibroblasts by ASCL1, microRNA-9/9*, and microRNA-124 Overexpression.

The common marmoset (Callithrix jacchus) has attracted considerable attention, especially in the biomedical science and neuroscience research fields, because of its potential to recapitulate the complex and multidimensional phenotypes of human diseases, and several neurodegenerative transgenic models have been reported. However, there remain several issues as (i) it takes years to generate late-onset disease models, and (ii) the onset age and severity of phenotypes can vary among individuals due to differences in genetic background. In the present study, we established an efficient and rapid direct neuronal induction method (induced neurons; iNs) from embryonic and adult marmoset fibroblasts to investigate cellular-level phenotypes in the marmoset brain in vitro. We overexpressed reprogramming effectors, i.e., microRNA-9/9*, microRNA-124, and Achaete-Scute family bHLH transcription factor 1, in fibroblasts with a small molecule cocktail that facilitates neuronal induction. The resultant iNs from embryonic and adult marmoset fibroblasts showed neuronal characteristics within two weeks, including neuron-specific gene expression and spontaneous neuronal activity. As directly reprogrammed neurons have been shown to model neurodegenerative disorders, the neuronal reprogramming of marmoset fibroblasts may offer new tools for investigating neurological phenotypes associated with disease progression in non-human primate neurological disease models.

MicroPhenoDB Associates Metagenomic Data with Pathogenic Microbes, Microbial Core Genes, and Human Disease Phenotypes

Microbes play important roles in human health and disease. The interaction between microbes and hosts is a reciprocal relationship, which remains largely under-explored. Current computational resources lack manually and consistently curated data to connect metagenomic data to pathogenic microbes, microbial core genes, and disease phenotypes. We developed the MicroPhenoDB database by manually curating and consistently integrating microbe-disease association data. MicroPhenoDB provides 5677 non-redundant associations between 1781 microbes and 542 human disease phenotypes across more than 22 human body sites. MicroPhenoDB also provides 696,934 relationships between 27,277 unique clade-specific core genes and 685 microbes. Disease phenotypes are classified and described using the Experimental Factor Ontology (EFO). A refined score model was developed to prioritize the associations based on evidential metrics. The sequence search option in MicroPhenoDB enables rapid identification of existing pathogenic microbes in samples without running the usual metagenomic data processing and assembly. MicroPhenoDB offers data browsing, searching, and visualization through user-friendly web interfaces and web service application programming interfaces. MicroPhenoDB is the first database platform to detail the relationships between pathogenic microbes, core genes, and disease phenotypes. It will accelerate metagenomic data analysis and assist studies in decoding microbes related to human diseases. MicroPhenoDB is available through http://www.liwzlab.cn/microphenodb and http://lilab2.sysu.edu.cn/microphenodb.

NcRNAVar: A Manually Curated Database for Identification of Noncoding RNA Variants Associated with Human Diseases

While variants of noncoding RNAs (ncRNAs) have been experimentally validated as a new class of biomarkers and drug targets, the discovery and interpretation of relationships between ncRNA variants and human diseases become important and challenging. Here we present ncRNAVar (http://www.liwzlab.cn/ncrnavar/), the first database that provides association data between validated ncRNA variants and human diseases through manual curation on 2650 publications and computational annotation. ncRNAVar contains 4565 associations between 711 human disease phenotypes and 3112 variants from 2597 ncRNAs. Each association was reviewed by professional curators, incorporated with valuable annotation and cross references, and designated with an association score by our refined score model. ncRNAVar offers web applications including association prioritization, network visualization, and relationship mapping. ncRNAVar, presenting a landscape of ncRNA variants in human diseases and a useful resource for subsequent software development, will improve our insight of relationships between ncRNA variants and human health.

Congenital ocular malformations in dogs and cats: 123 cases.

Provide epidemiological data regarding the prevalence of congenital ocular malformations in dogs and cats. A population of 32974 dogs and 13977 cats that presented for consultation at the veterinary teaching hospital. Medical records from 2011 to 2018 were reviewed. A retrospective and prospective epidemiological clinical study addressing congenital ocular malformations was conducted. Signalment, medical history, reason for presentation, clinical findings, vision impairment, and treatment options were analyzed. From the total of cases analyzed, 103 dogs (0.3%) and 20 cats (0.1%) met the inclusion criteria. The majority of dogs were mixed breed, the most common breed being the French Bulldog, while the majority of cats were European domestic shorthair. The median age of diagnosis was 12months for dogs and 6months for cats. Sex predisposition was not found. The most frequently identified abnormalities were as follows: congenital cataract (dogs: 31.1%; cats: 30.0%), microphthalmia (dogs: 35.0%, cats: 25.0%), and persistent pupillary membrane (dogs: 27.2%, cats: 40.0%). Some of the concurrently observed malformations were significantly associated. A statistically significant association was found between ocular dermoids and the French Bulldog breed (P<.001). Even though congenital ocular malformations are uncommon, knowledge about their prevalence is important, since they can cause vision impairment or even blindness. Moreover, some human ocular disease phenotypes are similar to the ones presented by dogs and cats, so they can be used as models to investigate pathophysiology and therapeutic approaches.

Genetic pathways disrupted by ENPP1 deficiency provide insight into mechanisms of osteoporosis, osteomalacia, and paradoxical mineralization.

Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.

Molecular Modeling of Pathogenic Mutations in the Keratin 1B Domain.

Keratin intermediate filaments constitute the primary cytoskeletal component of epithelial cells. Numerous human disease phenotypes related to keratin mutation remain mechanistically elusive. Our recent crystal structures of the helix 1B heterotetramer from keratin 1/10 enabled further investigation of the effect of pathologic 1B domain mutations on keratin structure. We used our highest resolution keratin 1B structure as a template for homology-modeling the 1B heterotetramers of keratin 5/14 (associated with blistering skin disorders), keratin 8/18 (associated with liver disease), and keratin 74/28 (associated with hair disorder). Each structure was examined for the molecular alterations caused by incorporating pathogenic 1B keratin mutations. Structural modeling indicated keratin 1B mutations can harm the heterodimer interface (R265PK5, L311RK5, R211PK14, I150VK18), the tetramer interface (F231LK1, F274SK74), or higher-order interactions needed for mature filament formation (S233LK1, L311RK5, Q169EK8, H128LK18). The biochemical changes included altered hydrophobic and electrostatic interactions, and altered surface charge, hydrophobicity or contour. Together, these findings advance the genotype-structurotype-phenotype correlation for keratin-based human diseases.

Population-scale proteome variation in human induced pluripotent stem cells.

Human disease phenotypes are driven primarily by alterations in protein expression and/or function. To date, relatively little is known about the variability of the human proteome in populations and how this relates to variability in mRNA expression and to disease loci. Here, we present the first comprehensive proteomic analysis of human induced pluripotent stem cells (iPSC), a key cell type for disease modelling, analysing 202 iPSC lines derived from 151 donors, with integrated transcriptome and genomic sequence data from the same lines. We characterised the major genetic and non-genetic determinants of proteome variation across iPSC lines and assessed key regulatory mechanisms affecting variation in protein abundance. We identified 654 protein quantitative trait loci (pQTLs) in iPSCs, including disease-linked variants in protein-coding sequences and variants with trans regulatory effects. These include pQTL linked to GWAS variants that cannot be detected at the mRNA level, highlighting the utility of dissecting pQTL at peptide level resolution.