Human Bronchial Epithelial Cell Transcriptome Changes in Response to Serum from Patients with Different Status of Inflammation.

PURPOSE: To investigate the transcriptome of human bronchial epithelial cells (HBEC) in response to serum from patients with different degrees of inflammation. METHODS: Serum from 19 COVID-19 patients obtained from the Hannover Unified Biobank was used. At the time of sampling, 5 patients had a WHO Clinical Progression Scale (WHO-CPS) score of 9 (severe illness). The remaining 14 patients had a WHO-CPS of below 9 (range 1-7), and lower illness. Multiplex immunoassay was used to assess serum inflammatory markers. The culture medium of HBEC was supplemented with 2% of the patient's serum, and the cells were cultured at 37 °C, 5% CO2 for 18 h. Subsequently, cellular RNA was used for RNA-Seq. RESULTS: Patients with scores below 9 had significantly lower albumin and serum levels of E-selectin, IL-8, and MCP-1 than patients with scores of 9. Principal component analysis based on 500 "core genes" of RNA-seq segregated cells into two subsets: exposed to serum from 4 (I) and 15 (II) patients. Cells from a subset (I) treated with serum from 4 patients with a score of 9 showed 5566 differentially expressed genes of which 2793 were up- and 2773 downregulated in comparison with cells of subset II treated with serum from 14 patients with scores between 1 and 7 and one with score = 9. In subset I cells, a higher expression of TLR4 and CXCL8 but a lower CDH1, ACE2, and HMOX1, and greater effects on genes involved in metabolic regulation, cytoskeletal organization, and kinase activity pathways were observed. CONCLUSION: This simple model could be useful to characterize patient serum and epithelial cell properties.

Acid Sphingomyelinase Deficiency Type B Patient-Derived Liver Organoids Reveals Altered Lysosomal Gene Expression and Lipid Homeostasis.

Acid sphingomyelinase deficiency (ASMD) or Niemann-Pick disease type A (NPA), type B (NPB) and type A/B (NPA/B), is a rare lysosomal storage disease characterized by progressive accumulation of sphingomyelin (SM) in the liver, lungs, bone marrow and, in severe cases, neurons. A disease model was established by generating liver organoids from a NPB patient carrying the p.Arg610del variant in the SMPD1 gene. Liver organoids were characterized by transcriptomic and lipidomic analysis. We observed altered lipid homeostasis in the patient-derived organoids showing the predictable increase in sphingomyelin (SM), together with cholesterol esters (CE) and triacylglycerides (TAG), and a reduction in phosphatidylcholine (PC) and cardiolipins (CL). Analysis of lysosomal gene expression pointed to 24 downregulated genes, including SMPD1, and 26 upregulated genes that reflect the lysosomal stress typical of the disease. Altered genes revealed reduced expression of enzymes that could be involved in the accumulation in the hepatocytes of sphyngoglycolipids and glycoproteins, as well as upregulated genes coding for different glycosidases and cathepsins. Lipidic and transcriptome changes support the use of hepatic organoids as ideal models for ASMD investigation.

Quantitative Lipid Profiling Reveals Major Differences between Liver Organoids with Normal Pi*M and Deficient Pi*Z Variants of Alpha-1-antitrypsin.

Different mutations in the SERPINA1 gene result in alpha-1 antitrypsin (AAT) deficiency and in an increased risk for the development of liver diseases. More than 90% of severe deficiency patients are homozygous for Z (Glu342Lys) mutation. This mutation causes Z-AAT polymerization and intrahepatic accumulation which can result in hepatic alterations leading to steatosis, fibrosis, cirrhosis, and/or hepatocarcinoma. We aimed to investigate lipid status in hepatocytes carrying Z and normal M alleles of the SERPINA1 gene. Hepatic organoids were developed to investigate lipid alterations. Lipid accumulation in HepG2 cells overexpressing Z-AAT, as well as in patient-derived hepatic organoids from Pi*MZ and Pi*ZZ individuals, was evaluated by Oil-Red staining in comparison to HepG2 cells expressing M-AAT and liver organoids from Pi*MM controls. Furthermore, mass spectrometry-based lipidomics analysis and transcriptomic profiling were assessed in Pi*MZ and Pi*ZZ organoids. HepG2 cells expressing Z-AAT and liver organoids from Pi*MZ and Pi*ZZ patients showed intracellular accumulation of AAT and high numbers of lipid droplets. These latter paralleled with augmented intrahepatic lipids, and in particular altered proportion of triglycerides, cholesterol esters, and cardiolipins. According to transcriptomic analysis, Pi*ZZ organoids possess many alterations in genes and cellular processes of lipid metabolism with a specific impact on the endoplasmic reticulum, mitochondria, and peroxisome dysfunction. Our data reveal a relationship between intrahepatic accumulation of Z-AAT and alterations in lipid homeostasis, which implies that liver organoids provide an excellent model to study liver diseases related to the mutation of the SERPINA1 gene.

Loss of Serpina1 in Mice Leads to Altered Gene Expression in Inflammatory and Metabolic Pathways.

The SERPINA1 gene encodes alpha1-antitrypsin (AAT), an acute phase glycoprotein and serine protease inhibitor that is mainly (80-90%) produced in the liver. Point mutations in the SERPINA1 gene can lead to the misfolding, intracellular accumulation, and deficiency of circulating AAT protein, increasing the risk of developing chronic liver diseases or chronic obstructive pulmonary disease. Currently, siRNA technology can knock down the SERPINA1 gene and limit defective AAT production. How this latter affects other liver genes is unknown. Livers were taken from age- and sex-matched C57BL/6 wild-type (WT) and Serpina1 knockout mice (KO) aged from 8 to 14 weeks, all lacking the five serpin A1a-e paralogues. Total RNA was isolated and RNA sequencing, and transcriptome analysis was performed. The knockout of the Serpina1 gene in mice changed inflammatory, lipid metabolism, and cholesterol metabolism-related gene expression in the liver. Independent single-cell sequencing data of WT mice verified the involvement of Serpina1 in cholesterol metabolism. Our results from mice livers suggested that designing therapeutic strategies for the knockout of the SERPINA1 gene in humans must account for potential perturbations of key metabolic pathways and consequent mitigation of side effects.

Differences in Expression of IQSEC2 Transcript Isoforms in Male and Female Cases with Loss of Function Variants and Neurodevelopmental Disorder.

Pathogenic hemizygous or heterozygous mutations in the IQSEC2 gene cause X-linked intellectual developmental disorder-1 (XLID1), characterized by a variable phenotype including developmental delay, intellectual disability, epilepsy, hypotonia, autism, microcephaly and stereotypies. It affects both males and females typically through loss of function in males and haploinsufficiency in heterozygous females. Females are generally less affected than males. Two novel unrelated cases, one male and one female, with de novo IQSEC2 variants were detected by trio-based whole exome sequencing. The female case had a previously undescribed frameshift mutation (NM_001111125:c.3300dup; p.Met1101Tyrfs*5), and the male showed an intronic variant in intron 6, with a previously unknown effect (NM_001111125:c.2459+21C>T). IQSEC2 gene expression study revealed that this intronic variant created an alternative donor splicing site and an aberrant product, with the inclusion of 19bp, confirming the pathogenic effect of the intron variant. Moreover, a strong reduction in the expression of the long, but also the short IQSEC2 isoforms, was detected in the male correlating with a more severe phenotype, while the female case showed no decreased expression of the short isoform, and milder effects of the disease. This suggests that the abnormal expression levels of the different IQSEC2 transcripts could be implicated in the severity of disease manifestations.

De novo small deletion affecting transcription start site of short isoform of AUTS2 gene in a patient with syndromic neurodevelopmental defects.

Disruption of the autism susceptibility candidate 2 (AUTS2) gene through genomic rearrangements, copy number variations (CNVs), and intragenic deletions and mutations, has been recurrently involved in syndromic forms of developmental delay and intellectual disability, known as AUTS2 syndrome. The AUTS2 gene plays an important role in regulation of neuronal migration, and when altered, associates with a variable phenotype from severely to mildly affected patients. The more severe phenotypes significantly correlate with the presence of defects affecting the C-terminus part of the gene. This article reports a new patient with a syndromic neurodevelopmental disorder, who presents a deletion of 30 nucleotides in the exon 9 of the AUTS2 gene. Importantly, this deletion includes the transcription start site for the AUTS2 short transcript isoform, which has an important role in brain development. Gene expression analysis of AUTS2 full-length and short isoforms revealed that the deletion found in this patient causes a remarkable reduction in the expression level, not only of the short isoform, but also of the full AUTS2 transcripts. This report adds more evidence for the role of mutated AUTS2 short transcripts in the development of a severe phenotype in the AUTS2 syndrome.

A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin.

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

New cis-Acting Variants in PI*S Background Produce Null Phenotypes Causing Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an inherited condition characterized by reduced levels of serum AAT due to mutations in the SERPINA1 (Serpin family A member 1) gene. The Pi*S (Glu264Val) is one of the most frequent deficient alleles of AATD, showing high incidence in the Iberian Peninsula. Herein, we describe two new alleles carrying an S mutation but producing a null phenotype: QOVigo and QOAachen. The new alleles were identified by sequencing the SERPINA1 gene in three patients who had lower AAT serum levels than expected for the initial genotype. These alleles are the result of combined mutations in cis in a PI*S allele. Sequencing detected the S mutation in cis with Tyr138Cys (S+Tyr138Cys) in two patients, whereas a third one had the S mutation in cis with Pro391Thr variant (S+Pro391Thr). When expressed in a cellular model, these variants caused strong AAT polymerization and very low AAT secretion to almost undetectable levels. The isoelectric focusing method for plasma AAT phenotyping did not show AAT protein encoded by the novel mutant alleles, behaving as null. We called these alleles PI*S-plus because the S variant was phased with another variant conferring more aggressive characteristics to the allele. The current data demonstrate that the clinical variability observed in AATD can be explained by additional genetic variation, such as dual cis-acting variants in the SERPINA1 gene. The possible existence of other unrevealed variants combined in the PI*S alleles should be considered to improve the genetic diagnosis of the patients.

Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Characterization of Novel Missense Variants of SERPINA1 Gene Causing Alpha-1 Antitrypsin Deficiency.

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

MicroRNA expression signatures for the prediction of BRCA1/2 mutation-associated hereditary breast cancer in paraffin-embedded formalin-fixed breast tumors.

Screening for germline mutations in breast cancer-associated genes BRCA1 and BRCA2 is indicated for patients with breast cancer from high-risk breast cancer families and influences both treatment options and clinical management. However, only 25% of selected patients test positive for BRCA1/2 mutation, indicating that additional diagnostic biomarkers are necessary. We analyzed 124 formalin-fixed paraffin-embedded (FFPE) tumor samples from patients with hereditary (104) and sporadic (20) invasive breast cancer, divided into two series (A and B). Microarray expression profiling of 829 human miRNAs was performed on 76 samples (Series A), and bioinformatics tool Prophet was used to develop and test a microarray classifier. Samples were stratified into a training set (n = 38) for microarray classifier generation and a test set (n = 38) for signature validation. A 35-miRNA microarray classifier was generated for the prediction of BRCA1/2 mutation status with a reported 95% (95% CI = 0.88-1.0) and 92% (95% CI: 0.84-1.0) accuracy in the training and the test set, respectively. Differential expression of 12 miRNAs between BRCA1/2 mutation carriers versus noncarriers was validated by qPCR in an independent tumor series B (n = 48). Logistic regression model based on the expression of six miRNAs (miR-142-3p, miR-505*, miR-1248, miR-181a-2*, miR-25* and miR-340*) discriminated between tumors from BRCA1/2 mutation carriers and noncarriers with 92% (95% CI: 0.84-0.99) accuracy. In conclusion, we identified miRNA expression signatures predictive of BRCA1/2 mutation status in routinely available FFPE breast tumor samples, which may be useful to complement current patient selection criteria for gene testing by identifying individuals with high likelihood of being BRCA1/2 mutation carriers.

Alternative transcripts of the SERPINA1 gene in alpha-1 antitrypsin deficiency.

BACKGROUND: SERPINA1 is the gene for alpha-1 antitrypsin (AAT), an acute phase protein with anti-protease and immunoregulatory activities. Mutations in SERPINA1 gene cause AAT deficiency and predispose individuals to early-onset emphysema and liver diseases. Expression of the SERPINA1 gene is regulated by different promoters and alternative splicing events among non-coding exons 1A, 1B and 1C. METHODS: We have developed three quantitative PCR (QT-PCR) assays (1A, 1B and 1C). These assays were applied for the analysis of SERPINA1 alternative transcripts in: (1) 16 human tissues and (2) peripheral blood leukocytes from 33 subjects with AAT mutations and 7 controls. RESULTS: Tissue-specific expression was found for the SERPINA1 transcripts. The 1A transcripts were mainly expressed in leukocytes and lung tissue while those detected with the 1B assay were highly restricted to leukocytes. Only 1B transcripts significantly correlated with serum AAT levels. The 1C transcripts were specifically found in lung, liver, kidney and pancreas. Furthermore, the expression of transcripts was related to AAT genotypes. While deficient variants of AAT had no pronounced effect on the transcript expression, null alleles were associated with significant reduction of different transcripts. CONCLUSIONS: The possibility to discriminate between SERPINA1 alternative splicing products will help us to understand better the regulation of SERPINA1 gene and its association with SERPINA1 mutations-related diseases.

Tumor microRNA expression profiling identifies circulating microRNAs for early breast cancer detection.

BACKGROUND: The identification of novel biomarkers for early breast cancer detection would be a great advance. Because of their role in tumorigenesis and stability in body fluids, microRNAs (miRNAs) are emerging as a promising diagnostic tool. Our aim was to identify miRNAs deregulated in breast tumors and evaluate the potential of circulating miRNAs in breast cancer detection. METHODS: We conducted miRNA expression profiling of 1919 human miRNAs in paraffin-embedded tissue from 122 breast tumors and 11 healthy breast tissue samples. Differential expression analysis was performed, and a microarray classifier was generated. The most relevant miRNAs were analyzed in plasma from 26 healthy individuals and 83 patients with breast cancer (36 before and 47 after treatment) and validated in 116 healthy individuals and 114 patients before treatment. RESULTS: We identified a large number of miRNAs deregulated in breast cancer and generated a 25-miRNA microarray classifier that discriminated breast tumors with high diagnostic sensitivity and specificity. Ten miRNAs were selected for further investigation, of which 4 (miR-505-5p, miR-125b-5p, miR-21-5p, and miR-96-5p) were significantly overexpressed in pretreated patients with breast cancer compared with healthy individuals in 2 different series of plasma. MiR-505-5p and miR-96-5p were the most valuable biomarkers (area under the curve 0.72). Moreover, the expression levels of miR-3656, miR-505-5p, and miR-21-5p were decreased in a group of treated patients. CONCLUSIONS: Circulating miRNAs reflect the presence of breast tumors. The identification of deregulated miRNAs in plasma of patients with breast cancer supports the use of circulating miRNAs as a method for early breast cancer detection.

Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid.

BACKGROUND: Severe Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. It is usually related to PI*Z alleles, and less frequent to rare and null (QO) alleles. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema. METHODS: A family with severe AAT deficiency was analyzed to achieve genetic diagnosis. The complete exons and introns of the SERPINA1 gene were sequenced and transcriptional analysis by RT-PCR was performed to characterize the effect of splicing variants found in the patients. In addition, a minigene MGserpa1_ex1b-1c was cloned into the pSAD vector to in vitro investigate the independent impact of variants on splicing process. RESULTS: We report a new identified null allele (PI*QOMadrid) in two adult siblings with practically no detectable serum AAT. The PI*QOMadrid allele consist of a duplication of the thymine (T) in position +2 of the donor splice site of exon 1C (+2dupT). In these two subjects, PI*QOMadrid occurred in compound heterozygote combination with the previously described variant PI*QOPorto. Both QOMadrid and QOPorto variants are located very close together in a regulatory region of the SERPINA1 gene. Analysis of transcripts revealed that QOMadrid variant prevented the expression of transcripts from exon 1C, and then normally spliced RNA products are not expected in the liver of these patients. In addition, aberrant splicing patterns of both variants were clearly distinguished and quantified by functional in vitro assays lending further support to their pathogenicity. CONCLUSION: Finding pathogenic mutations in non-coding regions of the SERPINA1 highlight the importance that regulatory regions might have in the disease. Regulatory regions should be seriously considered in discordant cases with severe AAT deficiency where no coding mutations were found.

MicroRNA profile in very young women with breast cancer.

BACKGROUND: Breast cancer is rarely diagnosed in very young women (35 years old or younger), and it often presents with distinct clinical-pathological features related to a more aggressive phenotype and worse prognosis when diagnosed at this early age. A pending question is whether breast cancer in very young women arises from the deregulation of different underlying mechanisms, something that will make this disease an entity differentiated from breast cancer diagnosed in older patients. METHODS: We performed a comprehensive study of miRNA expression using miRNA Affymetrix2.0 array on paraffin-embedded tumour tissue of 42 breast cancer patients 35 years old or younger, 17 patients between 45 and 65 years old and 29 older than 65 years. Data were statistically analyzed by t-test and a hierarchical clustering via average linkage method was conducted. Results were validated by qRT-PCR. Putative targeted pathways were obtained using DIANA miRPath online software. RESULTS: The results show a differential and unique miRNA expression profile of 121 miRNAs (p-value <0.05), 96 of those with a FDR-value <0.05. Hierarchical clustering grouped the samples according to their age, but not by subtype nor by tumour characteristics. We were able to validate by qRT-PCR differences in the expression of 6 miRNAs: miR-1228*, miR-3196, miR-1275, miR-92b, miR-139 and miR-1207. Moreover, all of the miRNAs maintained the expression trend. The validated miRNAs pointed out pathways related to cell motility, invasion and proliferation. CONCLUSIONS: The study suggests that breast cancer in very young women appears as a distinct molecular signature. To our knowledge, this is the first time that a validated microRNA profile, distinctive to breast cancer in very young women, has been presented. The miRNA signature may be relevant to open an important field of research in order to elucidate the underlying mechanism in this particular disease, which in a more clinical setting, could potentially help to identify therapeutic targets in this particular set of patients.

Genetic anticipation is associated with telomere shortening in hereditary breast cancer.

There is increasing evidence suggesting that short telomeres and subsequent genomic instability contribute to malignant transformation. Telomere shortening has been described as a mechanism to explain genetic anticipation in dyskeratosis congenita and Li-Fraumeni syndrome. Since genetic anticipation has been observed in familial breast cancer, we aimed to study telomere length in familial breast cancer patients and hypothesized that genetic defects causing this disease would affect telomere maintenance resulting in shortened telomeres. Here, we first investigated age anticipation in mother-daughter pairs with breast cancer in 623 breast cancer families, classified as BRCA1, BRCA2, and BRCAX. Moreover, we analyzed telomere length in DNA from peripheral blood leukocytes by quantitative PCR in a set of 198 hereditary breast cancer patients, and compared them with 267 control samples and 71 sporadic breast cancer patients. Changes in telomere length in mother-daughter pairs from breast cancer families and controls were also evaluated to address differences through generations. We demonstrated that short telomeres characterize hereditary but not sporadic breast cancer. We have defined a group of BRCAX families with short telomeres, suggesting that telomere maintenance genes might be susceptibility genes for breast cancer. Significantly, we described that progressive telomere shortening is associated with earlier onset of breast cancer in successive generations of affected families. Our results provide evidence that telomere shortening is associated with earlier age of cancer onset in successive generations, suggesting that it might be a mechanism of genetic anticipation in hereditary breast cancer.

An association between plasma levels of α2-macroglobulin and α1-antitrypsin in PiMM and PiZZ individuals differing in COPD presentation.

INTRODUCTION: Compared to normal PiMM, individuals with severe α1-antitrypsin (AAT) PiZZ (Glu342Lys) genotype deficiency are at higher risk of developing early-onset chronic obstructive pulmonary disease (COPD)/emphysema associated with Z-AAT polymers and neutrophilic inflammation. We aimed to investigate putative differences in plasma levels of acute phase proteins (APP) between PiMM and PiZZ subjects and to determine plasma Z-AAT polymer levels in PiZZ subjects. MATERIALS AND METHODS: Nephelometric analysis of seven plasma APPs was performed in 67 PiMM and 44 PiZZ subjects, of whom 43 and 42, respectively, had stable COPD. Of the PiZZ-COPD patients, 21 received and 23 did not receive intravenous therapy with human AAT preparations (IV-AAT). Plasma levels of Z-AAT polymers were determined by Western blotting using specific mouse monoclonal antibodies (2C1 and LG96). RESULTS: In addition to lower plasma AAT, PiZZ patients had higher α2-macroglobulin (A2MG) levels than PiMM patients. In contrast, PiZZ who received IV-AAT had higher AAT values but lower A2MG values than PiZZ without IV-AAT. Regardless of the AAT genotype, AAT levels were inversely correlated with A2MG, and the AAT/A2MG ratio was correlated with lung diffusion capacity (DCLO%). All PiZZ patients had circulating Z-AAT polymer levels that correlated directly with A2MG. In PiZZ without IV-AAT therapy polymer levels correlated inversely with the ratio of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC). CONCLUSION: Combined measurement of plasma AAT and A2MG levels may be of clinical value in assessing the progression of COPD and requires further attention.

Hyperkinetic Movement Disorder Caused by the Recurrent c.892C>T NACC1 Variant.

BACKGROUND: Genetic syndromes of hyperkinetic movement disorders associated with epileptic encephalopathy and intellectual disability are becoming increasingly recognized. Recently, a de novo heterozygous NACC1 (nucleus accumbens-associated 1) missense variant was described in a patient cohort including one patient with a combined mitochondrial oxidative phosphorylation (OXPHOS) deficiency. OBJECTIVES: The objective is to characterize the movement disorder in affected patients with the recurrent c.892C>T NACC1 variant and study the NACC1 protein and mitochondrial function at the cellular level. METHODS: The movement disorder was analyzed on four patients with the NACC1 c.892C>T (p.Arg298Trp) variant. Studies on NACC1 protein and mitochondrial function were performed on patient-derived fibroblasts. RESULTS: All patients had a generalized hyperkinetic movement disorder with chorea and dystonia, which occurred cyclically and during sleep. Complex I was found altered, whereas the other OXPHOS enzymes and the mitochondria network seemed intact in one patient. CONCLUSIONS: The movement disorder is a prominent feature of NACC1-related disease.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Short telomeres are frequent in hereditary breast tumors and are associated with high tumor grade.

Telomere shortening is a common event involved in malignant transformation. Critically short telomeres may trigger chromosomal aberrations and produce genomic instability leading to cancer development. Therefore, telomere shortening is a frequent molecular alteration in early stages of many epithelial tumors and in breast cancer correlates with stage and prognosis. A better understanding of the involvement of short telomeres in tumors may have a significant impact on patient management and the design of more specific treatments. To understand the role of telomere length (TL) in breast cancer etiology we measured the length of individual telomere signals in single cells by using quantitative telomere in situ hybridization in paraffin-embedded tissue from hereditary and sporadic breast cancers. A total of 104 tumor tissue samples from 75 familial breast tumors (BRCA1, n = 14; BRCA2, n = 13; non-BRCA1/2, n = 48) and 29 sporadic tumors were analyzed. Assessment of telomere signal intensity allowed estimation of the mean TL and related variables, such as percentage of critically short telomeres and percentage of cells with short telomeres. These data were correlated with the immunohistochemical expression of molecular breast cancer markers. Hereditary BRCA1, BRCA2, and non-BRCA1/2 tumors were characterized by shorter TL comparing to sporadic tumors. Considering all tumors, tumor grade was a strong risk factor determining the proportion of short telomeres or short telomere cells. Moreover, some histopathological features appeared to be differentially associated to hereditary or sporadic subgroups. Short telomeres correlated with ER-negative tumors in sporadic cases but not in familial cases, whereas a high level of apoptosis was associated with shorter telomeres in hereditary BRCA1 and BRCA2 tumors. In addition, TL helped to define a subset of non-BRCA1/2 tumors with short telomeres associated with increased expression of antiapoptotic proteins. These findings highlight the potential interest of TL measurements as markers of aggressiveness in breast cancer.