Human Variants Research Articles

Transcriptional network analysis of peripheral blood leukocyte subsets in multiple sclerosis identifies a pathogenic role for a cytotoxicity-associated gene network in myeloid cells.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting predominantly adults. It is a complex disease associated with both environmental and genetic risk factors. Although over 230 risk single-nucleotide polymorphisms have been associated with MS, all are common human variants. The mechanisms by which they increase the risk of MS, however, remain elusive. We hypothesized that a complex genetic phenotype such as MS could be driven by coordinated expression of genes controlled by transcriptional regulatory networks. We, therefore, constructed a gene coexpression network from microarray expression analyses of five purified peripheral blood leukocyte subsets of 76 patients with relapsing remitting MS and 104 healthy controls. These analyses identified a major network (or module) of expressed genes associated with MS that play key roles in cell-mediated cytotoxicity which was downregulated in monocytes of patients with MS. Manipulation of the module gene expression was achieved in vitro through small interfering RNA gene knockdown of identified drivers. In a mouse model, network gene knockdown modulated the autoimmune inflammatory MS model disease-experimental autoimmune encephalomyelitis. This research implicates a cytotoxicity-associated gene network in myeloid cells in the pathogenesis of MS.

Gain-of-function human UNC93B1 variants cause systemic lupus erythematosus and chilblain lupus.

UNC93B1 is a transmembrane domain protein mediating the signaling of endosomal Toll-like receptors (TLRs). We report five families harboring rare missense substitutions (I317M, G325C, L330R, R466S, and R525P) in UNC93B1 causing systemic lupus erythematosus (SLE) or chilblain lupus (CBL) as either autosomal dominant or autosomal recessive traits. As for a D34A mutation causing murine lupus, we recorded a gain of TLR7 and, to a lesser extent, TLR8 activity with the I317M (in vitro) and G325C (in vitro and ex vivo) variants in the context of SLE. Contrastingly, in three families segregating CBL, the L330R, R466S, and R525P variants were isomorphic with respect to TLR7 activity in vitro and, for R525P, ex vivo. Rather, these variants demonstrated a gain of TLR8 activity. We observed enhanced interaction of the G325C, L330R, and R466S variants with TLR8, but not the R525P substitution, indicating different disease mechanisms. Overall, these observations suggest that UNC93B1 mutations cause monogenic SLE or CBL due to differentially enhanced TLR7 and TLR8 signaling.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among these defects are recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-related mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, patients with FRM often experience worsening or demise following stress associated with infections. We investigated 2 female patients with FRM carrying the potentially novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from 3 patients with FRM fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

Transposase-assisted tagmentation: an economical and scalable strategy for single-worm whole-genome sequencing.

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in Caenorhabditis elegans, Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii. This method, demands merely 20 min of hands-on time for a single-worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the B-Raf Proto-oncogene, Serine/Threonine Kinase (BRAF) V600E mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

Human cytomegalovirus glycoprotein variants governing viral tropism and syncytium formation in epithelial cells and macrophages.

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.

Structure prediction analysis of human core TIM23 complex reveals conservation of the protein translocation mechanism.

The majority of mitochondrial proteins are encoded in the nucleus, translated on cytosolic ribosomes, and subsequently targeted to the mitochondrial surface. Their further import into the organelle is facilitated by highly specialized protein translocases. Mitochondrial precursor proteins that are destined to the mitochondrial matrix and, to some extent, the inner membrane, utilize translocase of the inner membrane (TIM23). This indispensable import machinery has been extensively studied in yeast. The translocating unit of the TIM23 complex in yeast consists of two membrane proteins, Tim17 and Tim23. In contrast to previous findings, recent reports demonstrate the primary role of Tim17, rather than Tim23, in the translocation of newly synthesized proteins. Very little is known about human TIM23 translocase. Human cells have two orthologs of yeast Tim17, TIMM17A and TIMM17B. Here, using computational tools, we present the architecture of human core TIM23 variants with either TIMM17A or TIMM17B, forming two populations of highly similar complexes. The structures reveal high conservation of the core TIM23 complex between human and yeast. Interestingly, both TIMM17A and TIMM17B variants interact with TIMM23 and reactive oxygen species modulator 1 (ROMO1); a homolog of yeast Mgr2, a protein that can create a channel-like structure with Tim17. The high structural conservation of proteins that form the core TIM23 complex in yeast and humans raises an interesting question about mechanistic and functional differences that justify existence of the two variants of TIM23 in higher eukaryotes.

Neuroinflammation and Lysosomal Abnormalities Characterise the Essential Role for Oxidation Resistance 1 in the Developing and Adult Cerebellum.

Loss-of-function mutations in the TLDc family of proteins cause a range of severe childhood-onset neurological disorders with common clinical features that include cerebellar neurodegeneration, ataxia and epilepsy. Of these proteins, oxidation resistance 1 (OXR1) has been implicated in multiple cellular pathways related to antioxidant function, transcriptional regulation and cellular survival; yet how this relates to the specific neuropathological features in disease remains unclear. Here, we investigate a range of loss-of-function mouse model systems and reveal that constitutive deletion of Oxr1 leads to a rapid and striking neuroinflammatory response prior to neurodegeneration that is associated with lysosomal pathology. We go on to show that neuroinflammation and cell death in Oxr1 knockouts can be completely rescued by the neuronal expression of Oxr1, suggesting that the phenotype is driven by the cell-intrinsic defects of neuronal cells lacking the gene. Next, we generate a ubiquitous, adult inducible knockout of Oxr1 that surprisingly displays rapid-onset ataxia and cerebellar neurodegeneration, establishing for the first time that the distinctive pathology associated with the loss of Oxr1 occurs irrespective of developmental stage. Finally, we describe two new homozygous human pathogenic variants in OXR1 that cause neurodevelopmental delay, including a novel stop-gain mutation. We also compare functionally two missense human pathogenic mutations in OXR1, including one newly described here, that cause different clinical phenotypes but demonstrate partially retained neuroprotective activity against oxidative stress. Together, these data highlight the essential role of Oxr1 in modulating neuroinflammatory and lysosomal pathways in the mammalian brain and support the hypothesis that OXR1 protein dosage may be critical for pathological outcomes in disease.

Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of β-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

Secretion of functional interferon by the type 3 secretion system of enteropathogenic Escherichia coli

BackgroundType I interferons (IFN-I)—a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties—are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells.ResultsIn this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN.ConclusionThese findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.

Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology.

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

Molecular Epidemiology of Human Norovirus Variants from Outbreaks in Zhejiang Province, China, during 2021

Molecular Epidemiology of Human Norovirus Variants from Outbreaks in Zhejiang Province, China, during 2021

Predicted mechanistic impacts of human protein missense variants.

Genome sequencing efforts have led to the discovery of tens of millions of protein missense variants found in the human population with the majority of these having no annotated role and some likely contributing to trait variation and disease. Sequence-based artificial intelligence approaches have become highly accurate at predicting variants that are detrimental to the function of proteins but they do not inform on mechanisms of disruption. Here we combined sequence and structure-based methods to perform proteome-wide prediction of deleterious variants with information on their impact on protein stability, protein-protein interactions and small-molecule binding pockets. AlphaFold2 structures were used to predict approximately 100,000 small-molecule binding pockets and stability changes for over 200 million variants. To inform on protein-protein interfaces we used AlphaFold2 to predict structures for nearly 500,000 protein complexes. We illustrate the value of mechanism-aware variant effect predictions to study the relation between protein stability and abundance and the structural properties of interfaces underlying trans protein quantitative trait loci (pQTLs). We characterised the distribution of mechanistic impacts of protein variants found in patients and experimentally studied example disease linked variants in FGFR1.

Mutation in PDGFRbeta: a potential new pathogenic variant for acquired mitral valve prolapse

Abstract Funding Acknowledgements None. Background Mitral valve prolapse (MVP) due to myxomatous degeneration is characterized by familial clustering, but few pathogenic genes have been identified. In a genetic screening program for MVP patients, we identified a platelet-derived growth factor receptor β (PDGFRβ)-E162K missense variant in a family with a bi-leaflet MVP phenotype in four affected family members. PDGFRβ is a receptor tyrosine kinase, which plays an important role in vascular development. Purpose In this study, we investigated whether the E162K substitution in PDGFRβ could lead to mitral valve abnormalities. Methods To assess the functional effects of the missense variant, proliferation and migration assays were performed using HeLa cells overexpressing wild-type (WT) or mutant PDGFRβ. Immunostainings were performed to determine PDGFRβ expression in the diseased valves from a family member undergoing surgical operation and from additional patients operated for MVP. Mouse models were created with the mouse equivalent of the human PDGFRβ-E162K variant (PDGFRβ-E161K) using CRISPR/CAS9. The variant was not lethal and transthoracic echocardiography was performed on adult homozygous PDGFRβE161K/E161K mice, heterozygous PDGFRβE161K/- mice, and WT mice . In addition, histology was performed in adult and neonatal mice to detect potential valvular defects. Results HeLa cells expressing PDGFRβ-E162K and stimulated with PDGFD showed reduced proliferation and migration, suggesting a loss of function of the mutant receptor. PDGFRβ was expressed in human diseased mitral valves. Echocardiographic evaluation revealed a significant larger mitral valve annulus diameter in both PDGFRβE161K/- and PDGFRβE161K/E161K compared to WT (WT: 1.97 mm; PDGFRβE161K/-:2.13, p=0.031; PDGFRβE161K/E161K:2.42 mm, p<0.0001). Histological data in PDGFRβE161K/E161K mice showed significant leaflet thickening in the free edge (FE) of the posterior mitral valve leaflet (PMVL) compared to WT (WT: 72.34 ɥm; PDGFRβE161K/E161K: 109.6 ɥm, p=0.040) and a larger leaflet area of the PMVL compared to WT (WT: 25280 ɥm²; PDGFRβE161K/E161K: 44494 ɥm², p=0.020). Moreover, an altered organization of the extracellular matrix reminiscent of the myxomatous degeneration in MVP was observed. These alterations were not observed in mutant neonatal hearts. Conclusions The PDGFRβ-E162K variant is associated with familial MVP and alters the function of PDGFRβ. Mice harboring this mutation display acquired mitral valve defects.

Zebrafish dspb-/- mutant as model for arrhythmogenic cardiomyopathy - impact of physical activity

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background Zebrafish has been reported as a model to study the Arrythmogenic cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. Purpose Our aim was the characterization of dspb-/- mutants, studying the impact of physical activity in development of the disease. Methods The characterization of phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) and adult (8 months old) individuals. The impact of physical activity was determined through moderated training periods (1 month) and endurance assays using a swimming tunnel. Results Viability assays showed that dspb mutants suffer a premature death, with significant higher mortality rates than the wildtype group (survival WT 89.7% vs dspb-/- 61.3%, p<0.01). Larvae cardiac function showed dspb mutants had a higher heart rate (WT 106.8 ± 26.8 vs dspb-/- 141.0 ± 7.2, p<0.01) while echocardiography showed differences in heart rate (WT 127.2 ± 49,8 vs dspb-/- 219.2 ± 26.8, p<0.05), systolic (WT 26.9 ± 5.3 vs dspb-/- 18.5 ± 5.2, p<0.05) and diastolic (WT 37.6 ± 8.4 vs dspb-/- 23.7 ± 5.4, p<0.05) areas. Gene expression analysis in adult heart showed dspb expression drastically decreased (WT 0.9 ± 0.1 vs dspb-/- 0.4 ± 0.01, p<0.01), whilst pkp2 expression increased (WT 0.9 ± 0.1 vs dspb-/- 2.59 ±0.22, p<0.001) as well as dspa gene expression (wt 0.9 ± 0.1 vs dspb-/- 4.41 ± 0.18, p< 0.001) as a compensation mechanism. Moderated long term physical activity had a positive impact in heart rate (dspb-/-pre 219.2 ± 26.8 vs dspb-/-post 138 ± 7.5, p<0.01) and diastolic area (dspb-/-pre 24 ± 5 vs dspb-/-post 32 ± 2.1, p<0.05). Adult mutants showed a better endurance showing improvement in immobile cumulative duration (dspb-/-pre 849.3 ± 29.3 vs dspb-/-post 179.8 ± 29.7, p<0.01) and immobile frequency (dspb-/-pre 5399.5 ± 1127.8 vs dspb-/-post 1310 ± 481.9, p<0.01). Conclusion The dspb-/- zebrafish presented pathological cardiac phenotype at larvae and adult stages, and less endurance in intensity exercises. The moderate exercise has turned out to be a beneficial modulator of the phenotype.

Zebrafish dspb -/- tert +/- mutant as model for arrhythmogenic cardiomyopathy. Impact of aging in the development of the disease

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background/Objectives Zebrafish has been reported as model to study the Arrythmogenic Cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic Cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. On the other hand, tert gene enables telomerase activity and telomere maintenance and mutations in this gene have been used for studying aging effects. After multiple crossings of our dspb model with tert mutants we have obtained dspb-/-tert+/- model line. Purpose Our aim was the characterization of dspb-/-tert+/- mutants, studying the impact of aging in the development of the disease. Methods The model was obtained by crossing dspb-/- and tert-/- mutants, crossing the heterozygous mutants and genotyping for the selection of the dspb-/-tert+/- mutants. The characterization of the phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) young adult (8 months old) and old adult (3 years old) individuals. Results Viability assays showed that dspb-/-tert+/- mutants suffer a premature death, with much lower survival rates than the dspb-/- group (survival dspb-/- 61.3% vs dspb-/-tert+/- 35.6%, p<0.01). Larvae heart rate analysis showed no significant differences. Echocardiography in young adults showed no significant differences between dspb-/-tert+/- and dspb-/- mutants. In old adults, echocardiography showed differences in heart rate (dspb-/- 142.5 ± 15.5 vs dspb-/-tert+/ -97.5 ± 3.5, p<0.01), respiratory frequency (dspb-/- 288.7 ± 36.1 vs dspb-/-tert+/- 95 ± 49.4, p<0.01) and atrial contraction velocity (dspb-/-155.7 ± 26.09 vs dspb-/-tert+/ -227.2 ± 17.6, p<0.05). Gene expression analysis in larvae showed that dspb expression drastically decreased (dspb-/- 0.3 ± 0.1 vs dspb-/-tert+/ -0.01 ± 0.008, p<0.01). In adults, dspb expression drastically decreased in heart tissue (dspb-/- 0.4 ± 0.01 vs dspb-/-tert+/- 0.06 ± 0.004, p<0.01) and skin (dspb-/ -0.34 ± 0.02 vs dspb-/-tert+/- 0.25 ± 0.01, p<0.01). Conclusion The dspb-/-tert+/- zebrafish presented worse survival rates, lower dspb gene expression levels and worse pathological cardiac phenotype at adult stages than dspb-/- mutants, proving that age is an important factor in the development of the disease and proving that dspb-/-tert+/- zebrafish is an appropriate model to study the impact of aging.

Ethnic Variation and Structure‐Function Analysis of Tauopathy‐Associated PERK Alleles

AbstractEIF2AK3, also known as PERK, plays a pivotal role in cellular proteostasis, orchestrating the Unfolded Protein Response (UPR) and Integrated Stress Response (ISR) pathways. In addition to its central position in intracellular stress regulation, human GWAS identify EIF2AK3 as a risk factor in tauopathies, neurodegenerative diseases caused by aberrant tau protein accumulation. Guided by these genomic indicators, our investigation systematically analyzed human PERK variants, focusing on those with potential tauopathy linkages. We assembled a comprehensive data set of human PERK variants associated with Wolcott Rallison Syndrome (WRS), tauopathies, and bioinformatically predicted loss‐of‐function, referencing the gnomAD, Ensembl, and NCBI databases. We found extensive racial/ethnic variation in the prevalence of common PERK polymorphisms linked to tauopathies. Using SWISS‐MODEL, we identified structural perturbations in the ER stress‐sensing luminal domain dimers/oligomers of tauopathy‐associated PERK variants, Haplotypes A and B, in combination with another tauopathy‐linked R240H mutation. Recombinant expression of disease‐associated variants in vitro revealed altered PERK signal transduction kinetics in response to ER stress compared to the predominant non‐disease variant. In summary, our data further substantiates that human PERK variants identified in tauopathy genetic studies negatively impact PERK structure, function, and downstream signaling with significant variations in prevalence among different racial and ethnic groups.

Expression of Truncated Products at the 5'-Terminal Region of RIPK2 and Evolutive Aspects that Support Their Biological Importance.

Alternative splicing is the process of generating different mRNAs from the same primary transcript, which contributes to increase the transcriptome and proteome diversity. Abnormal splicing has been associated with the development of several diseases including cancer. Given that mutations and abnormal levels of the RIPK2 transcript and RIP-2 protein are frequent in tumors, and that RIP-2 modulates immune and inflammatory responses, we investigated alternative splicing events that result in partial deletions of the kinase domain at the N-terminus of RIP-2. We also investigated the structure and expression of the RIPK2 truncated variants and isoforms in different environments. In addition, we searched data throughout Supraprimates evolution that could support the biological importance of RIPK2 alternatively spliced products. We observed that human variants and isoforms were differentially regulated following temperature stress, and that the truncated transcript was more expressed than the long transcript in tumor samples. The inverse was found for the longer protein isoform. The truncated variant was also detected in chimpanzee, gorilla, hare, pika, mouse, rat, and tree shrew. The fact that the same variant has been preserved in mammals with divergence times up to 70 million years raises the hypothesis that it may have a functional significance.

Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice

SIRT5 is a sirtuin deacylase that removes negatively charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal conditions, the phenotypes of SIRT5 deficiency are quite subtle. Here, we identify two homozygous SIRT5 variants in patients suspected to have mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generated a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology, or other gross phenotypes. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, but are likely not by themselves the primary pathogenic cause of the neuropathology observed in the patients.

Identification of the Molecular Components of Enhancer-Mediated Gene Expression Variation in Multiple Tissues Regulating Blood Pressure.

Inter-individual variation in blood pressure (BP) arises in part from sequence variants within enhancers modulating the expression of causal genes. We propose that these genes, active in tissues relevant to BP physiology, can be identified from tissue-level epigenomic data and genotypes of BP-phenotyped individuals. We used chromatin accessibility data from the heart, adrenal, kidney, and artery to identify cis-regulatory elements (CREs) in these tissues and estimate the impact of common human single-nucleotide variants within these CREs on gene expression, using machine learning methods. To identify causal genes, we performed a gene-wise association test. We conducted analyses in 2 separate large-scale cohorts: 77 822 individuals from the Genetic Epidemiology Research on Adult Health and Aging and 315 270 individuals from the UK Biobank. We identified 309, 259, 331, and 367 genes (false discovery rate <0.05) for diastolic BP and 191, 184, 204, and 204 genes for systolic BP in the artery, kidney, heart, and adrenal, respectively, in Genetic Epidemiology Research on Adult Health and Aging; 50% to 70% of these genes were replicated in the UK Biobank, significantly higher than the 12% to 15% expected by chance (P<0.0001). These results enabled tissue expression prediction of these 988 to 2875 putative BP genes in individuals of both cohorts to construct an expression polygenic score. This score explained ≈27% of the reported single-nucleotide variant heritability, substantially higher than expected from prior studies. Our work demonstrates the power of tissue-restricted comprehensive CRE analysis, followed by CRE-based expression prediction, for understanding BP regulation in relevant tissues and provides dual-modality supporting evidence, CRE and expression, for the causality genes.

Destabilization and Degradation of a Disease-Linked PGM1 Protein Variant.

PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.