LincRNA00612 inhibits apoptosis and inflammation in LPS-induced BEAS-2B cells via enhancing interaction between p-STAT3 and A2M promoter.

Long non-coding RNAs (lncRNAs) have been reported as key regulators of chronic obstructive pulmonary disease (COPD). This study aimed to figure out the regulatory mechanism as well as the effects of lncRNA00612 (LINC00612) in lipopolysaccharide (LPS)-induced inflammation and apoptosis in BEAS-2B cells. LINC00612 and its co-expressed gene alpha-2-macroglobulin (A2M) were strikingly downregulated in the peripheral venous blood of COPD patients. Overexpressed LINC00612 enhances BEAS-2B cells against apoptosis and inflammatory reactions mediated by LPS, however, an A2M knockdown can attenuate the degree of the enhancement. Bioinformatics analysis revealed putative binding sites between LINC00612, signal transducer and activator of transcription 3 (STAT3) and the A2M promoter, while RNA antisense purification and Chromatin immunoprecipitation were performed to confirm the prediction. Knockdown of LINC00612 impaired the binding of p-STAT3 to the promoter of A2M, which meant that LINC00612 was critical for the binding of STAT3 with the A2M promoter. Therefore, it can be concluded that LINC00612 ameliorates LPS-induced cell apoptosis and inflammation via recruiting STAT3 to bind to A2M. This conclusion will serve as a theoretical foundation for the treatment of COPD.

Audiologic Measures in an Indigenous Community with A2ML1- and FUT2-Related Otitis Media.

Background: Many indigenous peoples are at elevated risk for otitis media, however there is limited information on hearing loss due to OM in these communities. An Indigenous Filipino community that has previously been described with an elevated prevalence of OM that is due to rare A2ML1 variants and a common FUT2 variant underwent additional phenological testing. In this study, we describe the audiologic profiles in A2ML1- and FUT2-related otitis media and the validity of otoscopy and genotyping for A2ML1 and FUT2 variants in screening for otitis media and hearing loss. Method: We analyzed A2ML1 and FUT2 genotypes together with demographic, otologic and audiologic data from tympanometry and hearing level assessments of 109 indigenous individuals. Results: We confirmed previous findings of a spectrum of nonsyndromic otitis media as associated with A2ML1 variants. A2ML1 and FUT2 variants were associated with high-frequency hearing loss at 4000 Hz. As expected, young age was associated with flat tympanograms, and eardrum perforations due to chronic otitis media were associated with severe-to-profound hearing loss across frequencies. Adding A2ML1 or FUT2 genotypes improved the validity of otoscopy as a screening test to rule out moderate-to-profound hearing loss. Conclusion: Continued multi-disciplinary management and audiologic follow-up using tympanometry and screening audiometry are needed to document and treat otitis media and prevent permanent hearing loss in the indigenous community.

Overexpression of lncRNA A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/bone morphogenetic protein 3 axis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.

LncRNA A2M-AS1 Promotes Ferroptosis in Pancreatic Cancer via Interacting With PCBP3.

Ferroptosis is a newly-discovered cell death mechanism involved in the progression of various tumors, the role of noncoding RNAs (ncRNAs) in it was relatively less explored. This study identified the low levels of a recently studied long noncoding RNA (lncRNA), A2M-AS1, in pancreatic cancer and suggested its positive correlation with the overall survival time of patients with pancreatic cancer. A2M-AS1 was mainly localized in the cytoplasm, inhibiting the cellular proliferation, migration, and invasion as well as the tumor growth of the pancreatic cancer cells. Moreover, the Erastin-induced ferroptosis increased the expression levels of A2M-AS1. The overexpression of A2M-AS1 promoted ferroptosis in the pancreatic cancer, which was inhibited by the silencing of A2M-AS1. Mechanically, A2M-AS1 could directly interact with the poly (rC) binding protein 3 (PCBP3), which plays an important role in the process of iron metabolism, thereby promoting the ferroptosis in pancreatic cancer. In addition, the A2M-AS1/PCBP3 axis could facilitate the p38 activation and inhibit the phosphorylation of the AKT-mTOR signaling pathway; all these participate in regulating ferroptosis. In conclusion, the regulation of ferroptosis by targeting the A2M-AS1/PCBP3 axis might provide a novel target for the treatment of pancreatic cancer in the future. IMPLICATIONS: A2M-AS1 might be a potential novel therapeutic target for patients with pancreatic cancer in the future.

A2M is a potential core gene in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a type of malignant tumor ranking the second in the incidence of primary liver cancer following hepatocellular carcinoma. Both the morbidity and mortality have been increasing in recent years. Small duct type of ICC has potential therapeutic targets. But overall, the prognosis of patients with ICC is usually very poor. METHODS: To search latent therapeutic targets for ICC, we programmatically selected the five most suitable microarray datasets. Then, we made an analysis of these microarray datasets (GSE26566, GSE31370, GSE32958, GSE45001 and GSE76311) collected from the Gene Expression Omnibus (GEO) database. The GEO2R tool was effective to find out differentially expressed genes (DEGs) between ICC and normal tissue. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were executed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.8. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze protein-protein interaction of these DEGs and protein-protein interaction of these DEGs was modified by Cytoscape3.8.2. Survival analysis was performed using Gene Expression Profiling Interactive Analysis (GEPIA) online analysis tool. RESULTS: A total of 28 upregulated DEGs and 118 downregulated DEGs were screened out. Then twenty hub genes were selected according to the connectivity degree. The survival analysis results showed that A2M was closely related to the pathogenesis and prognosis of ICC and was a potential therapeutic target for ICC. CONCLUSIONS: According to our study, low A2M expression in ICC compared to normal bile duct tissue was an adverse prognostic factor in ICC patients. The value of A2M in the treatment of ICC needs to be further studied.

Association Between Common Variants of APOE, ABCA7, A2M, BACE1, and Cerebrospinal Fluid Biomarkers in Alzheimer's Disease: Data from the PUMCH Dementia Cohort.

BACKGROUND: The previous studies have identified several genes in relation to Alzheimer's disease (AD), such as ABCA7, CR1, etc. A few studies have explored the association between the common variants, mainly in the non-coding regions of these genes, and cerebrospinal fluid (CSF) biomarkers. Fewer studies target the variants in the coding regions. OBJECTIVE: To illustrate the association between the common variants within or adjacent to the coding regions of AD susceptible genes and CSF biomarkers in AD patients. METHODS: 75 sporadic probable AD patients were extracted from the dementia cohort of Peking Union Medical College Hospital. They all had history inquiry, physical examination, blood test, cognitive assessment, brain MRI, CSF testing of Aß42, 181p-tau, and t-tau, and next-generation DNA sequencing. Sixty-nine common single nucleotide polymorphisms (SNPs) (minor allele frequency > 0.01) within or near the coding region of 13 AD susceptible genes were included in the analysis. RESULTS: The rs7412-CC (APOE) genotype showed lower CSF Aß42 level and higher p-tau/Aß42 ratio than the rs7412-CT genotype. The rs3752246-C (ABCA7) allele correlated with lower CSF Aß42 level. The alternate alleles of six ABCA7 SNPs were related to lower CSF p-tau, including rs3745842, rs3764648, rs3764652, rs4147930, rs4147934 and rs881768. The rs11609582-TT (A2M) genotype showed higher CSF p-tau than the rs11609582-TA genotype. The p-tau/Aß42 ratio was higher in the rs490460-TT (BACE1) genotype relative to the rs490460-GT genotype. CONCLUSION: Some common variants within or near the coding regions of APOE, ABCA7, A2M, and BACE1 are associated with CSF Aß42, p-tau. or p-tau/Aß42.

Biallelic variants in CPAMD8 are associated with primary open-angle glaucoma and primary angle-closure glaucoma.

BACKGROUND/AIMS: This study aims to assess the contribution of biallelic CPAMD8 variants in patients with different forms of glaucoma, especially primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG), based on a systematic analysis of exome sequencing (ES). METHODS: Potentially pathogenic CPAMD8 variants were selected from the ES data of 5307 subjects with various eye conditions through multiple bioinformatics analyses. Of the 5307 subjects, 1221 probands had different forms of primary glaucoma. The genotype-phenotype correlation was assessed by a systematic review of biallelic CPAMD8 variants that including our data and data from the literature. The expression profile of CPAMD8 in human tissues was determined at the mRNA and protein levels. RESULTS: Biallelic CPAMD8 variants, including one frameshift and six missense variants, were exclusively present and significantly enriched in patients with glaucoma (one with juvenile open-angle glaucoma (JOAG), two with POAG and two with PACG) compared with none of the 4086 probands with other eye conditions in this cohort (p=4.1E-07). The effect of variants in these patients is relatively mild compared with that reported in patients with anterior segment dysgenesis or primary congenital glaucoma. CPAMD8 mRNA was highly expressed in the optic nerve, ciliary body, retina and iris, whereas the CPAMD8 protein was mainly detected in the nonpigmented epithelium of the iris and ciliary process, determined by immunohistochemistry. CONCLUSIONS: The data from this study not only provide further evidence to support the association of biallelic CPAMD8 variants with JOAG but also suggest that biallelic CPAMD8 variants might be associated with POAG and PACG.

Indomethacin Disrupts the Formation of ß-Amyloid Plaques via an α2-Macroglobulin-Activating lrp1-Dependent Mechanism.

Epidemiological studies have implied that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the development and progression of Alzheimer's disease (AD). However, the underlying mechanisms are notably understudied. Using a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) (APP/PS1) expressing transgenic (Tg) mice and neuroblastoma (N) 2a cells as in vivo and in vitro models, we revealed the mechanisms of indomethacin in ameliorating the cognitive decline of AD. By screening AD-associated genes, we observed that a marked increase in the expression of α2-macroglobulin (A2M) was markedly induced after treatment with indomethacin. Mechanistically, upregulation of A2M was caused by the inhibition of cyclooxygenase-2 (COX-2) and lipocalin-type prostaglandin D synthase (L-PGDS), which are responsible for the synthesis of prostaglandin (PG)H2 and PGD2, respectively. The reduction in PGD2 levels induced by indomethacin alleviated the suppression of A2M expression through a PGD2 receptor 2 (CRTH2)-dependent mechanism. Highly activated A2M not only disrupted the production and aggregation of ß-amyloid protein (Aß) but also induced Aß efflux from the brain. More interestingly, indomethacin decreased the degradation of the A2M receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which facilitated the brain efflux of Aß. Through the aforementioned mechanisms, indomethacin ameliorated cognitive decline in APP/PS1 Tg mice by decreasing Aß production and clearing Aß from the brains of AD mice.

Effects of ethanolic extracts of Quercus, Cirsium vulgare, and Falcaria vulgaris on gastric ulcer, antioxidant and inflammatory indices, and gene expression in rats.

INTRODUCTION: Gastric ulcer is a multifaceted process and is usually caused by mucosal damage. Herbal medicines have received much attention considering the side effects of chemical drugs. Nowadays, the use of herbal medicines has received much attention considering the side effects of chemical drugs. Quercus brantii Lindl, Cirsium vulgare (Savi) Ten, and Falcaria vulgaris Bernh are plants used as traditional phytomedicine for gastric ulcer diseases. AIM OF THE STUDY: This study was aimed to investigate the protective effects of hydroalcoholic extracts of these herbs on ethanol-induced gastric ulceration, in addition, to investigate the antioxidant, anti-inflammatory, and gene expression. MATERIALS AND METHODS: Thirty Sprague Dawley rats, (200-250 g), were divided into six groups: Control: intact animals; sham: gavaged with distilled water (14 days); negative control: gavaged with 20 mg/kg of omeprazole (14 days); experimental groups I, II, and III: gavaged with 500 mg/kg of the extract of Falcaria vulgaris, Quercus brantii, and Cirsium vulgare, respectively, (14 days). The number of ulcers and pathological parameters were assessed. The serum superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, total antioxidant capacity, albumin, total protein, haptoglobin, alpha-1-acid glycoprotein, total globulin, alpha-2-macroglobulin, C-fos, C-myc, and Caspase-9 were measured by ELISA and RT-PCR. RESULTS: The extracts significantly reduced gastric ulcer (52.33%). The results showed that the Quercus brantii extract was more effective. There were significant differences between the serum levels of alpha-1-acid glycoprotein and those of alpha-2-macroglobulin. Also, there was a significant difference in the serum level of antioxidant parameters. Changes in the expression of the genes also confirmed the results suggested by other parameters. The expression levels of C-fos, C-myc, and caspase-9 were decreased, but the Bcl-2 expression increased. CONCLUSION: The hydro-alcoholic extracts revealed various protection and noticeable change in the expression of caspase-9, C-myc, C-fos, and Bcl-2 genes in rats.

Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism.

Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking-mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region's position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M's protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

Identification of 5S and 45S rDNA sites in Chrysanthemum species by using oligonucleotide fluorescence in situ hybridization (Oligo-FISH).

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

The clinical significance of A2ML1 variants in Noonan syndrome has to be reconsidered.

The RASopathies are a group of clinically and genetically heterogeneous developmental disorders caused by dysregulation of the RAS/MAPK signalling pathway. Variants in several components and regulators of this pathway have been identified as the pathogenetic cause. In 2015, missense variants in A2ML1 were reported in three unrelated families with clinical diagnosis of Noonan syndrome (NS) and a zebrafish model was presented showing heart and craniofacial defects similar to those caused by a NS-associated Shp2 variant. However, a causal role of A2ML1 variants in NS has not been confirmed since. Herein, we report on 15 individuals who underwent screening of RASopathy-associated genes and were found to carry rare variants in A2ML1, including variants previously proposed to be causative for NS. In cases where parental DNA was available, the respective A2ML1 variant was found to be inherited from an unaffected parent. Seven index patients carrying an A2ML1 variant presented with an alternate disease-causing genetic aberration. These findings underscore that current evidence is insufficient to support a causal relation between variants in A2ML1 and NS, questioning the inclusion of A2ML1 screening in diagnostic RASopathy testing.

Substituting the Thiol Ester of Human A2M or C3 with a Disulfide Produces Native Proteins with Altered Proteolysis-Induced Conformational Changes.

Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H2O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Epigenetic Modulation by Apabetalone Counters Cytokine-Driven Acute Phase Response In Vitro, in Mice and in Patients with Cardiovascular Disease.

Chronic systemic inflammation contributes to cardiovascular disease (CVD) and correlates with the abundance of acute phase response (APR) proteins in the liver and plasma. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate inflammatory gene transcription. We show that BET inhibition by the small molecule apabetalone reduces APR gene and protein expression in human hepatocytes, mouse models, and plasma from CVD patients. Steady-state expression of serum amyloid P, plasminogen activator inhibitor 1, and ceruloplasmin, APR proteins linked to CVD risk, is reduced by apabetalone in cultured hepatocytes and in humanized mouse liver. In cytokine-stimulated hepatocytes, apabetalone reduces the expression of C-reactive protein (CRP), alpha-2-macroglobulin, and serum amyloid P. The latter two are also reduced by apabetalone in the liver of endotoxemic mice. BET knockdown in vitro also counters cytokine-mediated induction of the CRP gene. Mechanistically, apabetalone reduces the cytokine-driven increase in BRD4 BET occupancy at the CRP promoter, confirming that transcription of CRP is BET-dependent. In patients with stable coronary disease, plasma APR proteins CRP, IL-1 receptor antagonist, and fibrinogen γ decrease after apabetalone treatment versus placebo, resulting in a predicted downregulation of the APR pathway and cytokine targets. We conclude that CRP and components of the APR pathway are regulated by BET proteins and that apabetalone counters chronic cytokine signaling in patients.

MFGE8, ALB, APOB, APOE, SAA1, A2M, and C3 as Novel Biomarkers for Stress Cardiomyopathy.

BACKGROUND: Stress cardiomyopathy (SCM) is a transient reversible left ventricular dysfunction that more often occurs in women. Symptoms of SCM patients are similar to those of acute coronary syndrome (ACS), but little is known about biomarkers. The goals of this study were to identify the potentially crucial genes and pathways associated with SCM. METHODS: We analyzed microarray datasets GSE95368 derived from the Gene Expression Omnibus (GEO) database. Firstly, identify the differentially expressed genes (DEGs) between SCM patients in normal patients. Then, the DEGs were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, the protein-protein interaction (PPI) network was constructed and Cytoscape was used to find the key genes. RESULTS: In total, 25 DEGs were identified, including 10 upregulated genes and 15 downregulated genes. These DEGs were mainly enriched in ECM-receptor interaction, dilated cardiomyopathy (DCM), human papillomavirus infection, and focal adhesion, whereas in GO function classification, they were mainly enriched in the extracellular region, positive regulation of the multicellular organismal process, establishment of localization, and intracellular vesicle. CONCLUSION: Seven hub genes contained APOE, MFGE8, ALB, APOB, SAA1, A2M, and C3 identified as hub genes of SCM, which might be used as diagnostic biomarkers or molecular targets for the treatment of SCM.

Digenic inheritance of subclinical variants in Noonan Syndrome patients: an alternative pathogenic model?

Noonan syndrome (NS) is an autosomal-dominant disorder with variable expressivity and locus heterogeneity. Despite several RAS pathway genes were implicated in NS, 20-30% of patients remain without molecular diagnosis, suggesting the involvement of further genes or multiple mechanisms. Eight patients out of 60, negative for conventional NS mutation analysis, with heterogeneous NS phenotype were investigated by means of target resequencing of 26 RAS/MAPK pathway genes. A trio was further characterized by means of whole-exome sequencing. Protein modeling and in silico prediction of protein stability allowed to identify possible pathogenic RAS pathway variants in four NS patients. A new c.355T>C variant in LZTR1 was found in patient 43. Two patients co-inherited variants in LRP1 and LZTR1 (patient 53), or LRP1 and SOS1 genes (patient 67). The forth patient (56) carried a compound heterozygote of RASAL3 gene variants and also an A2ML1 variant. While these subclinical variants are singularly present in healthy parents, they co-segregate in patients, suggesting their addictive effect and supporting a digenic inheritance, as alternative model to a more common monogenic transmission. The ERK1/2 and SAPK/JNK activation state, assessed on immortalized lymphocytes from patients 53 and 67 showed highest phosphorylation levels compared to their asymptomatic parents. These findings together with the lack of their co-occurrence in the 1000Genomes database strengthen the hypothesis of digenic inheritance in a subset of NS patients. This study suggests caution in the exclusion of subclinical variants that might play a pathogenic role providing new insights for alternative hereditary mechanisms.

Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion.

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

Detection of autoantibodies against alpha-2-macroglobulin-like 1 in paraneoplastic pemphigus sera utilizing novel green fluorescent protein-based immunoassays.

BACKGROUND: Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies. OBJECTIVES: To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera. METHODS: We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates. RESULTS: A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values. CONCLUSIONS: Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens.

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.