Observational studies provide evidence that metabolites may be involved in the development of autoimmune diseases (ADs), but whether it is causal is still unknown. Based on the large-scale genome-wide association studies (GWAS) summary statistics, we performed two-sample Mendelian randomization (MR) to evaluate the causal associations between human blood metabolites and multiple ADs, which were inflammatory bowel disease (IBD), ulcerative colitis (UC), crohns disease (CD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), multiple sclerosis (MS), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). After Bonferroni adjustment, we identified 6 causal features of metabolites, i.e., glycerol 2-phosphate for T1D, hexadecanedioate, phenylacetylglutamine and laurylcarnitine for RA, glycine and arachidonate (20:4n6) for CD. Comprehensive sensitive analysis was further performed to validate the robustness of associations. We also observed some overlaps of metabolites among different ADs, implying similar or shared underlying mechanisms in such pathogenic processes. Multivariable MR analysis was then conducted to avoid potential pleiotropic effect of other complex traits. After controlling for several common traits, multivariable MR analysis ruled out most of potential pleiotropic effects and validated independence of identified metabolites. Finally, metabolic pathway analysis was performed based on suggestive metabolites for each AD respectively and a total of seven metabolic pathways were identified. In conclusion, this study provided novel insights into investigating causal role of blood metabolites in development of multiple ADs through a comprehensive genetic pathway.

Recent advances in next-generation sequencing, deep networks and other bioinformatic tools have enabled us to mine huge amount of genomic information about living organisms in the post-microarray era. However, these tools do not explicitly factor in the role of the underlying DNA biochemistry (particularly, DNA hybridization) essential to life processes. Here, we focus more precisely on the role that DNA hybridization plays in determining properties of biological organisms at the macro-level. We illustrate its role with solutions to challenging problems in human disease. These solutions are made possible by novel structural properties of DNA hybridization landscapes revealed by a metric model of oligonucleotides of a common length that makes them reminiscent of some planets in our solar system, particularly Earth and Saturn. They allow a judicious selection of so-called noncrosshybridizing (nxh) bases that offer substantial reduction of DNA sequences of arbitrary length into a few informative features. The quality assessment of the information extracted by them is high because of their very low Shannon Entropy, i.e. they minimize the degree of uncertainty in hybridization that makes results on standard microarrays irreproducible. For example, SNP classification (pathogenic/non-pathogenic) and pathogen identification can be solved with high sensitivity (~77%/100%) and specificity (~92%/100%, respectively) for combined taxa on a sample of over 264 fully coding sequences in whole bacterial genomes and fungal mitochondrial genomes using machine learning (ML) models. These methods can be applied to several other interesting research questions that could be addressed with similar genomic analyses.

TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. In addition, we document seven novel TARS2 variants-one nonsense variant and six missense variants-that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modeling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.

GNAO1 encephalopathy is a neurodevelopmental disorder with a spectrum of symptoms that include dystonic movements, seizures and developmental delay. While numerous GNAO1 mutations are associated with this disorder, the functional consequences of pathological variants are not completely understood. Here, we deployed the invertebrate C. elegans as a whole-animal behavioral model to study the functional effects of GNAO1 disorder-associated mutations. We tested several pathological GNAO1 mutations for effects on locomotor behaviors using a combination of CRISPR/Cas9 gene editing and transgenic overexpression in vivo. We report that all three mutations tested (G42R, G203R and R209C) result in strong loss of function defects when evaluated as homozygous CRISPR alleles. In addition, mutations produced dominant negative effects assessed using both heterozygous CRISPR alleles and transgenic overexpression. Experiments in mice confirmed dominant negative effects of GNAO1 G42R, which impaired numerous motor behaviors. Thus, GNAO1 pathological mutations result in conserved functional outcomes across animal models. Our study further establishes the molecular genetic basis of GNAO1 encephalopathy, and develops a CRISPR-based pipeline for functionally evaluating mutations associated with neurodevelopmental disorders.

The environment and events that we are exposed to in utero, during birth and in early childhood influence our future physical and mental health. The underlying mechanisms that lead to these outcomes are unclear, but long-term changes in epigenetic marks, such as DNA methylation, could act as a mediating factor or biomarker. DNA methylation data were assayed at 713 522 CpG sites from 9537 participants of the Generation Scotland: Scottish Family Health Study, a family-based cohort with extensive genetic, medical, family history and lifestyle information. Methylome-wide association studies of eight early life environment phenotypes and two adult mental health phenotypes (major depressive disorder and brief resilience scale) were conducted using DNA methylation data collected from adult whole blood samples. Two genes involved with different developmental pathways (PRICKLE2, Prickle Planar Cell Polarity Protein 2 and ABI1, Abl-Interactor-1) were annotated to CpG sites associated with preterm birth (P < 1.27 × 10-9). A further two genes important to the development of sensory pathways (SOBP, Sine Oculis Binding Protein Homolog and RPGRIP1, Retinitis Pigmentosa GTPase Regulator Interacting Protein) were annotated to sites associated with low birth weight (P < 4.35 × 10-8). The examination of methylation profile scores and genes and gene-sets annotated from associated CpGs sites found no evidence of overlap between the early life environment and mental health conditions. Birth date was associated with a significant difference in estimated lymphocyte and neutrophil counts. Previous studies have shown that early life environments influence the risk of developing mental health disorders later in life; however, this study found no evidence that this is mediated by stable changes to the methylome detectable in peripheral blood.

Several pharmacogenetics studies have identified an association between a greater metformin-dependent reduction in HbA1c levels and the minor A allele at rs2289669 in intron 10 of SLC47A1, encoding multidrug and toxin extrusion 1 (MATE1), a presumed metformin transporter. It is currently unknown if the rs2289669 locus is a cis-eQTL, which would validate its role as predictor of metformin efficacy. We looked at association between common genetic variants in the SLC47A1 gene region and HbA1c reduction after metformin treatment using locus-wise meta-analysis from the MetGen consortium. CRISPR-Cas9 was applied to perform allele editing of, or genomic deletion around, rs2289669 and of the closely linked rs8065082 in HepG2 cells. The genome-edited cells were evaluated for SLC47A1 expression and splicing. None of the common variants including rs2289669 showed significant association with metformin response. Genomic editing of either rs2289669 or rs8065082 did not alter SLC47A1 expression or splicing. Experimental and in silico analyses show that the rs2289669-containing haploblock does not appear to carry genetic variants that could explain its previously reported association with metformin efficacy.

Individuals with Phelan-McDermid syndrome (PMS) present with a wide range of developmental, medical, cognitive and behavioral abnormalities. Previous literature has begun to elucidate genotype-phenotype associations that may contribute to the wide spectrum of features. Here, we report results of genotype-phenotype associations in a cohort of 170 individuals with PMS. Genotypes were defined as Class I deletions (including SHANK3 only or SHANK3 with ARSA and/or ACR and RABL2B), Class II deletions (all other deletions) or sequence variants. Phenotype data were derived prospectively from direct evaluation, caregiver interview and questionnaires, and medical history. Analyses revealed individuals with Class I deletions or sequence variants had fewer delayed developmental milestones and higher cognitive ability compared to those with Class II deletions but had more skill regressions. Individuals with Class II deletions were more likely to have a variety of medical features, including renal abnormalities, spine abnormalities, and ataxic gait. Those with Class I deletions or sequence variants were more likely to have psychiatric diagnoses including bipolar disorder, depression, and schizophrenia. Autism spectrum disorder diagnoses did not differ between groups. This study represents the largest and most rigorous genotype-phenotype analysis in PMS to date and provides important information for considering clinical functioning, trajectories and comorbidities as a function of specific genetic alteration.

SHQ1 is essential for biogenesis of H/ACA ribonucleoproteins, a class of molecules important for processing ribosomal RNAs, modifying spliceosomal small nuclear RNAs and stabilizing telomerase. Components of the H/ACA ribonucleoprotein complex have been linked to neurological developmental defects. Here, we report two sibling pairs from unrelated families with compound heterozygous variants in SHQ1. Exome sequencing was used to detect disease causing variants, which were submitted to 'matching' platforms linked to MatchMaker Exchange. Phenotype comparisons supported these matches. The affected individuals present with early-onset dystonia, with individuals from one family displaying additional neurological phenotypes, including neurodegeneration. As a result of cerebrospinal fluid studies suggesting possible abnormal dopamine metabolism, a trial of levodopa replacement therapy was started but no clear response was noted. We show that fibroblasts from affected individuals have dramatic loss of SHQ1 protein. Variants from both families were expressed in Saccharomyces cerevisiae, resulting in a strong reduction in H/ACA snoRNA production and remarkable defects in rRNA processing and ribosome formation. Our study identifies SHQ1 as associated with neurological disease, including early-onset dystonia, and begins to delineate the molecular etiology of this novel condition.

Limb-girdle muscular dystrophy R3 (LGMDR3) is caused by mutations in the SGCA gene coding for α-sarcoglycan (SG). Together with β- γ- and δ-SG, α-SG forms a tetramer embedded in the dystrophin associated protein complex crucial for protecting the sarcolemma from mechanical stresses elicited by muscle contraction. Most LGMDR3 cases are due to missense mutations, which result in non-properly folded, even though potentially functional α-SG. These mutants are prematurely discarded by the cell quality control. Lacking one subunit, the SG-complex is disrupted. The resulting loss of function leads to sarcolemma instability, muscle fiber damage and progressive limb muscle weakness. LGMDR3 is severely disabling and, unfortunately, still incurable. Here, we propose the use of small molecules, belonging to the class of cystic fibrosis transmembrane regulator (CFTR) correctors, for recovering mutants of α-SG defective in folding and trafficking. Specifically, CFTR corrector C17 successfully rerouted the SG-complex containing the human R98H-α-SG to the sarcolemma of hind-limb muscles of a novel LGMDR3 murine model. Notably, the muscle force of the treated model animals was fully recovered. To our knowledge, this is the first time that a compound designated for cystic fibrosis is successfully tested in a muscular dystrophy and may represent a novel paradigm of treatment for LGMDR3 as well as different other indications in which a potentially functional protein is prematurely discarded as folding-defective. Furthermore, the use of small molecules for recovering the endogenous mutated SG has an evident advantage over complex procedures such as gene or cell transfer.

The metabolic needs for postnatal growth of the human nervous system are vast. Recessive loss-of-function mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms in neurons required for neuronal growth and survival. These metabolic processes include neuronal alanine synthesis and anaplerosis, the replenishment of tricarboxylic acid (TCA) cycle intermediates. We performed metabolomics across postnatal development in Gpt2-null mouse brain to identify the trajectory of dysregulated metabolic pathways: alterations in alanine occur earliest; followed by reduced TCA cycle intermediates and reduced pyruvate; followed by elevations in glycolytic intermediates and amino acids. Neuron-specific deletion of GPT2 in mice is sufficient to cause motor abnormalities and death pre-weaning, a phenotype identical to the germline Gpt2-null mouse. Alanine biosynthesis is profoundly impeded in Gpt2-null neurons. Exogenous alanine is necessary for Gpt2-null neuronal survival in vitro but is not needed for Gpt2-null astrocytes. Dietary alanine supplementation in Gpt2-null mice enhances animal survival and improves the metabolic profile of Gpt2-null brain but does not alone appear to correct motor function. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons in vivo. We also observe selective death of lower motor neurons in vivo with worsening motor behavior with age. In conclusion, these studies of the pathophysiology of GPT2 Deficiency have identified metabolic mechanisms that are required for neuronal growth and that potentially underlie selective neuronal vulnerabilities in motor neurons.

Intellectual disability (ID) is a neurodevelopmental disorder affecting approximately 0.5-3% of the population in the developed world. Individuals with ID exhibit deficits in intelligence, impaired adaptive behavior and often visual impairments. Cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2) is an interacting partner of the FMR protein, whose loss results in fragile X syndrome, the most common inherited cause of ID. Recently, CYFIP2 variants have been found in patients with early-onset epileptic encephalopathy, developmental delay and ID. Such individuals often exhibit visual impairments; however, the underlying mechanism is poorly understood. In the present study, we investigated the role of Cyfip2 in retinal and visual functions by generating and analyzing Cyfip2 conditional knockout (CKO) mice. While we found no major differences in the layer structures and cell compositions between the control and Cyfip2 CKO retinas, a subset of genes associated with the transporter and channel activities was differentially expressed in Cyfip2 CKO retinas than in the controls. Multi-electrode array recordings showed more sustained and stronger responses to positive flashes of the ON ganglion cells in the Cyfip2 CKO retina than in the controls, although electroretinogram analysis revealed that Cyfip2 deficiency unaffected the photoreceptor and ON bipolar cell functions. Furthermore, analysis of initial and late phase optokinetic responses demonstrated that Cyfip2 deficiency impaired the visual function at the organismal level. Together, our results shed light on the molecular mechanism underlying the visual impairments observed in individuals with CYFIP2 variants and, more generally, in patients with neurodevelopmental disorders, including ID.

Germline-activating mutations in HRAS cause Costello syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here, we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via reactive oxygen species-dependent AMP-activated protein kinase α and p38 hyperactivation, occurs in primary fibroblasts of CS patients, resulting in accelerated glycolysis and increased fatty acid synthesis and storage as lipid droplets. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.

The retinal pigment epithelium of the vertebrate eyes acquires vitamin A from circulating retinol binding protein for chromophore biosynthesis. The chromophore covalently links with an opsin protein in the adjacent photoreceptors of the retina to form the bipartite visual pigment complexes. We here analyzed visual pigment biosynthesis in mice deficient for the retinol-binding protein receptor STRA6. We observed that chromophore content was decreased throughout the life cycle of these animals, indicating that lipoprotein-dependent delivery pathways for the vitamin cannot substitute for STRA6. Changes in the expression of photoreceptor marker genes, including a downregulation of the genes encoding rod and cone opsins, paralleled the decrease in ocular retinoid concentration in STRA6-deficient mice. Despite this adaptation, cone photoreceptors displayed absent or mislocalized opsins at all ages examined. Rod photoreceptors entrapped the available chromophore but exhibited significant amounts of chromophore-free opsins in the dark-adapted stage. Treatment of mice with pharmacological doses of vitamin A ameliorated the rod phenotype but did not restore visual pigment synthesis in cone photoreceptors of STRA6-deficient mice. The imbalance between chromophore and opsin concentrations of rod and cone photoreceptors was associated with an unfavorable retinal physiology, including diminished electrical responses of photoreceptors to light, and retinal degeneration during aging. Together, our study demonstrates that STRA6 is critical to adjust the stoichiometry of chromophore and opsins in rod and cone photoreceptors and to prevent pathologies associated with ocular vitamin A deprivation.

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs), thereby aggravating the airway wall remodeling during asthma; however, the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS + si-CRNDE (a siRNA targets long non-coding RNA CRNDE), respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO, and cell viability, proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE), inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically, CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKKβ phosphorylation, thereby activating the nuclear factor kappa B (NF-κB) pathway. Functionally, silencing CRNDE in LPS-EXO, an IKKβ inhibitor, and an NF-κB inhibitor all removed the upregulation of cell viability, proliferation and migration induced by LPS-EXO in ASMCs. In the end, the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-κB pathway by enhancing IKKβ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma.