De novo EIF2AK1 and EIF2AK2 Variants Are Associated with Developmental Delay, Leukoencephalopathy, and Neurologic Decompensation.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

An ultrasensitive electrochemical/colorimetric dual-mode self-powered biosensing platform for lung cancer marker detection by multiple-signal amplification strategy.

BACKGROUND: In recent years, miRNAs have emerged as potentially valuable tumor markers, and their sensitive and accurate detection is crucial for early screening and diagnosis of tumors. However, the analysis of miRNAs faces significant challenges due to their short sequence, susceptibility to degradation, high similarity, low expression level in cells, and stringent requirements for in vitro research environments. Therefore, the development of sensitive and efficient new methods for the detection of tumor markers is crucial for the early intervention of related tumors. RESULTS: An ultrasensitive electrochemical/colorimetric dual-mode self-powered biosensor platform is established to detect microRNA-21 (miR-21) via a multi-signal amplification strategy. Gold nanoparticles (AuNPs) and VS4 nanosheets self-assembled 3D nanorods (VS4-Ns-Nrs) are prepared for constructing a superior performance enzyme biofuel cell (EBFC). The double-signal amplification strategy of Y-shaped DNA nanostructure and catalytic hairpin assembly (CHA) is adopted to further improve enhance the strength and specificity of the output signal. In addition, a capacitor is matched with EBFC to generate an instantaneous current that is amplified several times, and the output detection signal is improved once more. At the same time, electrochemical and colorimetric methods are used for dual-mode strategy to achieve the accuracy of detection. The linear range of detection is from 0.001 pg/mL to 1000 pg/mL, with a relatively low limit of detection (LOD) of 0.16 fg/mL (S/N = 3). SIGNIFICANCE: The established method enables accurate and sensitive detection of markers in patients with lung cancer, providing technical support and data reference for precise identification. It is anticipated to offer a sensitive and practical new technology and approach for early diagnosis, clinical treatment, and drug screening of cancer and other related major diseases.

AI-MARRVEL - A Knowledge-Driven AI System for Diagnosing Mendelian Disorders.

BACKGROUND: Diagnosing genetic disorders requires extensive manual curation and interpretation of candidate variants, a labor-intensive task even for trained geneticists. Although artificial intelligence (AI) shows promise in aiding these diagnoses, existing AI tools have only achieved moderate success for primary diagnosis. METHODS: AI-MARRVEL (AIM) uses a random-forest machine-learning classifier trained on over 3.5 million variants from thousands of diagnosed cases. AIM additionally incorporates expert-engineered features into training to recapitulate the intricate decision-making processes in molecular diagnosis. The online version of AIM is available at https://ai.marrvel.org. To evaluate AIM, we benchmarked it with diagnosed patients from three independent cohorts. RESULTS: AIM improved the rate of accurate genetic diagnosis, doubling the number of solved cases as compared with benchmarked methods, across three distinct real-world cohorts. To better identify diagnosable cases from the unsolved pools accumulated over time, we designed a confidence metric on which AIM achieved a precision rate of 98% and identified 57% of diagnosable cases out of a collection of 871 cases. Furthermore, AIM's performance improved after being fine-tuned for targeted settings including recessive disorders and trio analysis. Finally, AIM demonstrated potential for novel disease gene discovery by correctly predicting two newly reported disease genes from the Undiagnosed Diseases Network. CONCLUSIONS: AIM achieved superior accuracy compared with existing methods for genetic diagnosis. We anticipate that this tool may aid in primary diagnosis, reanalysis of unsolved cases, and the discovery of novel disease genes. (Funded by the NIH Common Fund and others.).

Correlation of serum CA199 levels with glycemic control and microvascular complications in patients with type 2 diabetes mellitus.

OBJECTIVE: This study aimed to explore the correlation between glycemic control, microvascular complications and serum glycogen antigen (CA199) in patients with type 2 diabetes mellitus (T2DM). METHODS: 519 patients with T2DM admitted to our hospital were included. All patients had CA199 levels measured. Patients were divided into low glycation (LH) group (HbA1C <7.5%), Hyperglycemia (HH) group (HbA1C ≥7.5%), fasting glucose compliance (SF) group (FBG <7.0 mmol/L), high fasting glucose (HF) group (FBG ≥7.0 mmol/L), postprandial glucose compliance (SP) group (PBG <10.0 mmol/L) and high postprandial glucose (HP) group (PBG ≥10.0 mmol/L) and with microvascular complications group, and no microvascular complications group. Division was according to levels of glycated hemoglobin (HbA1C), fasting blood glucose (FBG), 2-hour postprandial blood glucose (2hPBG), and diabetic microvascular complications. RESULTS: CA199 levels were significantly higher in the HH and HF groups than in the LH and SF groups (P<0.05); HbA1C and FBG were positively correlated with CA199; CA199 levels were not significantly different between SP and HP groups (P>0.05), and PBG was not significantly correlated with CA199 levels. CA199 levels were significantly higher in the group with microvascular complications than in the group without microvascular complications (P<0.05); HbA1C was an independent risk factor for elevated CA199. CONCLUSION: Patients with T2DM and higher CA199 levels need to be evaluated for glycemic status and the presence of microvascular complications. HbA1C is a major risk factor for elevated CA199 levels.

Effect of Cyclocarya paliurus polysaccharides on streptozotocin-induced diabetic nephropathy in rats.

OBJEVTIVE: To investigate the efficacy of Cyclocarya paliurus (C. paliurus) polysaccharides on stre- ptozotocin-induced diabetic nephropathy in rats. METHODS: Rats were divided into 6 groups, including group of normal control, group of diabetic control, group of metformin treatment, low-dose group of C. paliurus polysaccharides treatment, middle-dose group of C. paliurus polysaccharides treatment and high-dose group of C. paliurus polysaccharides treatment. Histological analysis of kidney was analyzed using hematoxilin and eosin. Levels of blood glucose, creatinine, urea, uric acid were determined by spectrophotometry. Anti-oxidative enzymes were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Advanced glycation end products (AGEs) and transforming growth factor-ß1 (TGF-ß1) level was measured by ELISA. RESULTS: Abnormal changes were observed in the group of diabetic control characterized by atrophy of the renal glomeruli with hypercellularity, congestion of glomerular tufts, dilation of the renal spaces, and degeneration of renal tubule. Compared with that of normal group, blood glucose, creatinine, urea, uric acid level was significantly increased in the group of diabetic control. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase level was significantly decreased, but AGEs and TGF-ß1 level was significantly increased. By contrast, administration of C. paliurus polysaccharides and metformin could reverse the above-mentioned results of the group of diabetic control, especially in the high-dose group of C. paliurus polysaccharides. CONCLUSION: Our findings suggest that C. paliurus polysaccharides may play a protecting role for nephropathy of diabetic rats by lowering glucose, creatinine, urea, uric acid level, enhancing the antioxidative ability, and reducing AGEs and TGF-ß1 expression.

Loss of proteins associated with amyotrophic lateral sclerosis affects lysosomal acidification via different routes.

Abnormal accumulation of proteins is a hallmark of a variety of neurological diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Maintenance of protein homeostasis (proteostasis) in neurons via proteasomal and macroautophagy/autophagy-lysosomal degradation is thought to be central for proper neuronal function and survival. We recently reported evolutionarily conserved roles for two ALS-linked proteins, UBQLN2 (ubiquilin 2) and VAPB, in regulation of lysosomal degradation. Ubiquilins are required for v-ATPase-mediated lysosomal acidification, whereas VAPs are required for the PtdIns4P-mediated endo-lysosomal trafficking pathway.

Using MARRVEL v1.2 for Bioinformatics Analysis of Human Genes and Variant Pathogenicity.

One of the greatest challenges in the bioinformatic analysis of human sequencing data is identifying which variants are pathogenic. Numerous databases and tools have been generated to address this difficulty. However, these many useful data and tools are broadly dispersed, requiring users to search for their variants of interest through human genetic databases, variant function prediction tools, and model organism databases. To solve this problem, we collected data and observed workflows of human geneticists, clinicians, and model organism researchers to carefully select and display valuable information that facilitates the evaluation of whether a variant is likely to be pathogenic. This program, Model organism Aggregated Resources for Rare Variant ExpLoration (MARRVEL) v1.2, allows users to collect relevant data from 27 public sources for further efficient bioinformatic analysis of the pathogenicity of human variants. © 2019 by John Wiley & Sons, Inc.

Ubiquilins regulate autophagic flux through mTOR signalling and lysosomal acidification.

Although the aetiology of amyotrophic lateral sclerosis (ALS) remains poorly understood, impaired proteostasis is a common feature of different forms of ALS. Mutations in genes encoding ubiquilins, UBQLN2 and UBQLN4, cause familial ALS. The role of ubiquilins in proteasomal degradation is well established, but their role in autophagy-lysosomal clearance is poorly defined. Here, we describe a crosstalk between endoplasmic reticulum stress, mTOR signalling and autophagic flux in Drosophila and mammalian cells lacking ubiquilins. We found that loss of ubiquilins leads to endoplasmic reticulum stress, impairs mTORC1 activity, promotes autophagy and causes the demise of neurons. We show that ubiquilin mutants display defective autophagic flux due to reduced lysosome acidification. Ubiquilins are required to maintain proper levels of the V0a/V100 subunit of the vacuolar H+-ATPase and lysosomal pH. Feeding flies acidic nanoparticles alleviates defective autophagic flux in ubiquilin mutants. Hence, our studies reveal a conserved role for ubiquilins as regulators of autophagy by controlling vacuolar H+-ATPase activity and mTOR signalling.

VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway.

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. We find that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. We found that loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. We propose that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation. Abbreviations: ALS: amyotrophic lateral sclerosis; CSF: cerebrospinal fluid; CERT: ceramide transfer protein; FFAT: two phenylalanines in an acidic tract; MSP: major sperm proteins; OSBP: oxysterol binding protein; PH: pleckstrin homology; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns4K: phosphatidylinositol 4-kinase; UPR: unfolded protein response; VAMP: vesicle-associated membrane protein; VAPA/B: mammalian VAPA and VAPB proteins; VAPs: VAMP-associated proteins (referring to Drosophila Vap33, and human VAPA and VAPB).

Phospholipase PLA2G6, a Parkinsonism-Associated Gene, Affects Vps26 and Vps35, Retromer Function, and Ceramide Levels, Similar to α-Synuclein Gain.

Mutations in PLA2G6 (PARK14) cause neurodegenerative disorders in humans, including autosomal recessive neuroaxonal dystrophy and early-onset parkinsonism. We show that loss of iPLA2-VIA, the fly homolog of PLA2G6, reduces lifespan, impairs synaptic transmission, and causes neurodegeneration. Phospholipases typically hydrolyze glycerol phospholipids, but loss of iPLA2-VIA does not affect the phospholipid composition of brain tissue but rather causes an elevation in ceramides. Reducing ceramides with drugs, including myriocin or desipramine, alleviates lysosomal stress and suppresses neurodegeneration. iPLA2-VIA binds the retromer subunits Vps35 and Vps26 and enhances retromer function to promote protein and lipid recycling. Loss of iPLA2-VIA impairs retromer function, leading to a progressive increase in ceramide. This induces a positive feedback loop that affects membrane fluidity and impairs retromer function and neuronal function. Similar defects are observed upon loss of vps26 or vps35 or overexpression of α-synuclein, indicating that these defects may be common in Parkinson disease.

The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation.

Organisms have evolved elaborate mechanisms to adjust intracellular nutrient levels in response to fluctuating availability of exogenous nutrients. During starvation, cells can enhance amino acid uptake and synthesis through the general amino acid control (GAAC) pathway, whereas nonessential cellular contents are recycled by autophagy. How these two pathways are coordinated in response to starvation is currently unknown. Here we show that the GAAC pathway couples exogenous amino acid availability with autophagy. Starvation caused deactivation of mTOR, which then activated autophagy. In parallel, serum/glutamine starvation activated the GAAC pathway, which up-regulated amino acid transporters, leading to increased amino acid uptake. This elevated the intracellular amino acid level, which in turn reactivated mTOR and suppressed autophagy. Knockdown of activating transcription factor 4, the major transcription factor in the GAAC pathway, or of SLC7A5, a leucine transporter, caused impaired mTOR reactivation and much higher levels of autophagy. Thus, the GAAC pathway modulates autophagy by regulating amino acid uptake and mTOR reactivation during serum/glutamine starvation.

Mitochondria-localized glutamic acid-rich protein (MGARP) gene transcription is regulated by Sp1.

BACKGROUND: Mitochondria-localized glutamic acid-rich protein (MGARP) is a novel mitochondrial transmembrane protein expressed mainly in steroidogenic tissues and in the visual system. Previous studies showed that MGARP functions in hormone biosynthesis and its expression is modulated by the HPG axis. METHODOLOGY/PRINCIPAL FINDINGS: By bioinformatics, we identified two characteristic GC-rich motifs that are located proximal to the transcription start site (TSS) of MGARP, and each contains two Specificity protein 1 (Sp1) binding elements. We then determined that the -3 kb proximal MGARP promoter is activated in a Sp1-dependent manner using reporter assays and knockdown of Sp1 led to decreased expression of endogenous MGARP messages. We also demonstrated that one of the two GC-rich motifs, GC-Box1, harbors prominent promoter activity mediated by Sp1, and that it requires both GC boxes for full transcriptional activation. These findings suggest a dominant role for these GC boxes and Sp1 in activating the MGARP promoter through a synergistic mechanism. Consistently, the results of an Electrophoretic Mobility Gel Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) confirmed that Sp1 specifically interacts with the GC-rich region. We further found that estrogen receptor α (ERα), a known Sp1 co-activator, could potentiate GC-boxes containing MGARP promoter activity and this effect is mediated by Sp1. Knockdown of Sp1 significantly diminished the MGARP promoter transactivation and the expression of endogenous MGARP mediated by both Sp1 and ERα. CONCLUSIONS/SIGNIFICANCE: The present study identified a proximal core sequence in the MGARP promoter that is composed of two enriched Sp1 binding motifs and established Sp1 as one major MGARP transactivator whose functions are synergistic with ERα, providing a novel understanding of the mechanisms of MGARP gene transcriptional regulation.