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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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% CO 2 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., (© 2024. The Author(s).)

Plasma levels of α 1 -antitrypsin-derived C-terminal peptides might be valid as novel biomarkers to predict and/or characterise exacerbations in PiMM and PiZZ COPD patients, or to reflect the efficiency of augmentation therapy in PiZZ patients https://bit.ly/3rNJeLd., Competing Interests: Conflict of interest: T. Welte reports support for the present manuscript from the German Ministry of Education and Research, and payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from Grifols and CSL Behring, outside the submitted work. Conflict of interest: J. Chorostowska-Wynimko reports grants or contracts from AstraZeneca, Pfizer, CSL Behring, Grifols and Mereo Biopharma, outside the submitted work; consulting fees from CSL Behring, Grifols, Mereo Biopharma, Amgen and Pfizer, outside the submitted work; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from AstraZeneca, MSD, Pfizer, Takeda, Amgen, Grifols, CSL Behring, Novartis, Chiesi, Celon Pharma and Adamed, outside the submitted work; support for attending meetings and/or travel from MSD, Amgen and Pfizer, outside the submitted work; participation on a data safety monitoring or advisory board for CSL Behring, Grifols and Mereo Biopharma, outside the submitted work; leadership or fiduciary roles in other boards, societies, committees or advocacy groups, paid or unpaid, for the European Respiratory Society, Polish Respiratory Society, International Respiratory Coalition, Polish Coalition for Respiratory Disorders, Polish Coalition for Treatment of Asthma and Polish Foundation for Patients with Alpha-1 Antitrypsin Deficiency, outside the submitted work; and receipt of equipment, materials, drugs, medical writing, gifts or other services from Roche, Biocartis, Amoy, CSL Behring, and Pfizer, outside the submitted work. Conflict of interest: M. Kiehntopf reports that he is inventor of a patent covering a method for quantification of C-terminal peptides of AAT (applicant: Jena University Hospital (JUH); EP22154836.5; status:application), and the inventor of other published patents covering C-terminal AAT peptides in inflammation (applicant: Jena University Hospital (JUH): Method for determining the origin of an infection (EP17719610.2 (application); EP16167699.4 (granted)) and Diagnosis of Sepsis and Systemic Inflammatory Response Syndrome (CN104204808B, EP2592421, EP2780719, US10712350B2, JP6308946B2 (all granted)). Conflict of interest: S. Janciauskiene reports support for the present manuscript from German Center for Lung Research DZL; payment to her institution for research from Excellgene SA and Monthey, Switzerland, outside the submitted work; speaker at the Alpha1 expert meeting, Austria, for Chiesi GmbH, outside the submitted work; support for attending meetings from CSL Behring, outside the submitted work; and is a scientific advisor for the Alpha1 Patient Association, Germany, outside the submitted work. Conflict of interest: The remaining author have nothing to disclose., (Copyright ©The authors 2023.)

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.

Rodent models of lipopolysaccharide (LPS)-induced pulmonary inflammation are used for anti-inflammatory drug testing. We aimed to characterize mice responses to aerosolized LPS alone or with intraperitoneal (i.p.) delivery of alpha1-antitrypsin (AAT). Balb/c mice were exposed to clean air or aerosolized LPS (0.21 mg/mL) for 10 min per day, for 3 d. One hour after each challenge, animals were treated i.p. with saline or with (4 mg/kg body weight) one of the AAT preparations: native (AAT), oxidized (oxAAT), recombinant (recAAT), or peptide of AAT (C-36). Experiments were terminated 6 h after the last dose of AATs. Transcriptome data of mice lungs exposed to clean air versus LPS revealed 656 differentially expressed genes and 155 significant gene ontology terms, including neutrophil migration and toll-like receptor signaling pathways. Concordantly, mice inhaling LPS showed higher bronchoalveolar lavage fluid neutrophil counts and levels of myeloperoxidase, inducible nitric oxide synthase, IL-1β, TNFα, KC, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Plasma inflammatory markers did not increase. After i.p. application of AATs, about 1% to 2% of proteins reached the lungs but, except for GM-CSF, none of the proteins significantly influenced inflammatory markers. All AATs and C-36 significantly inhibited LPS-induced GM-CSF release. Surprisingly, only oxAAT decreased the expression of several LPS-induced inflammatory genes, such as Cxcl3, Cd14, Il1b, Nfkb1, and Nfkb2, in lung tissues. According to lung transcriptome data, oxAAT mostly affected genes related to transcriptional regulation while native AAT or recAAT affected genes of inflammatory pathways. Hence, we present a feasible mice model of local lung inflammation induced via aerosolized LPS that can be useful for systemic drug testing., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To explore the relationship between cancer cell SREBF1 expression, lipid droplets (LDs) formation, and the sensitivity to chemotherapies, we cultured lung adenocarcinoma cells H1299 (with LD) and H1563 (without LD) in a serum-free basal medium (BM) or neutrophil degranulation products containing medium (NDM), and tested cell responses to cisplatin and etoposide. By using the DESeq2 Bioconductor package, we detected 674 differentially expressed genes (DEGs) associated with NDM/BM differences between two cell lines, many of these genes were associated with the regulation of sterol and cholesterol biosynthesis processes. Specifically, SREBF1 markedly declined in both cell lines cultured in NDM or when treated with chemotherapeutics. Despite the latter, H1563 exhibited LD formation and resistance to etoposide, but not to cisplatin. Although H1299 cells preserved LDs, these cells were similarly sensitive to both drugs. In a cohort of 292 patients with non-small-cell lung cancer, a lower SREBF1 expression in tumors than in adjacent nontumor tissue correlated with overall better survival, specifically in patients with adenocarcinoma at stage I. Our findings imply that a direct correlation between SREBF1 and LD accumulation can be lost due to the changes in cancer cell environment and/or chemotherapy. The role of LDs in lung cancer development and response to therapies remains to be examined in more detail.

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.

Rare diseases affect more than 300 million people worldwide. Diagnosing rare diseases is a major challenge as they have different causes and etiologies. Careful assessment of clinical symptoms often leads to the testing of the most common genetic alterations that could explain the disease. Patients with negative results for these tests frequently undergo whole exome or genome sequencing, leading to the identification of the molecular cause of the disease in 50% of patients at best. Therefore, a significant proportion of patients remain undiagnosed after sequencing their genome. Recently, approaches based on functional aspects of the genome, including transcriptomics and epigenomics, are beginning to emerge. Here, we will review these approaches, including studies that have successfully provided diagnoses for complex undiagnosed cases.

Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host's misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation., Competing Interests: ST, MB, SW, MK, ML, RL, BM, EK, DD, NJ, MF, SJ No competing interests declared, TW reports grants from German Ministry of Research and Education, during the conduct of the study; personal fees from Grifols, CSL Behring, outside the submitted work, PS reports grants and personal fees from CSL Behring, grants and personal fees from Grifols Inc, personal fees from Dicerna Inc, grants from Vertex, grants from Arrowhead, outside the submitted work, JS reports grants from CSL Behring, grants from Kamada LtD, during the conduct of the study, (© 2021, Tumpara et al.)

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.

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: QO Vigo and QO Aachen . 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.

Human α1-antitrypsin (AAT) is an abundant acute phase glycoprotein expressing anti-protease and immunomodulatory activities, and is used as a biopharmaceutical to treat patients with inherited AAT deficiency. The pleiotropic properties of AAT provide a rationale for using this therapy outside of inherited AAT deficiency. Therapy with AAT is administrated intravenously, yet the alternative routes are being considered. To examine the putative transepidermal application of AAT we used epiCS®, the 3D human epidermis equivalents reconstructed from human primary epidermal keratinocytes. We topically applied various concentrations of AAT protein with a constant volume of 50 µl, prepared in Hank's balance solution, HBSS, to epiCS cultured under bas\al condition or when culture medium supplemented with 100 µg/ml of a combined bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) mixture. AAT freely diffused across epidermis layers in a concentration and time-dependent manner. Within 18 h topically provided 0.2 mg AAT penetrated well the stratum corneum and localizes within the keratinocytes. The treatments with AAT did not induce obvious morphological changes and damages in keratinocyte layers. As expected, LPS/PGN triggered a strong pro-inflammatory activation of epiCS. AAT exhibited a limited capacity to neutralize the effect of LPS/PGN, but more importantly, it lowered expression of IL-18 and IL-8, and preserved levels of filaggrin, a key protein for maintaining the epidermal barrier integrity. Our findings suggest that the transepidermal route for delivering AAT is worthwhile to explore further. If successful, this approach may offer an easy-to-use therapy with AAT for skin inflammatory diseases., (Copyright © 2020 Tumpara, Martinez-Delgado, Gomez-Mariano, Liu, DeLuca, Korenbaum, Jonigk, Jugert, Wurm, Wurm, Welte and Janciauskiene.)

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.

Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (© 2014 UICC.)

Hereditary breast cancer constitutes only 5-10% of all breast cancer cases and is characterized by strong family history of breast and/or other associated cancer types. Only ~ 25% of hereditary breast cancer cases carry a mutation in BRCA1 or BRCA2 gene, while mutations in other rare high and moderate-risk genes and common low penetrance variants may account for additional 20% of the cases. Thus the majority of cases are still unaccounted for and designated as BRCAX tumors. MicroRNAs are small non-coding RNAs that play important roles as regulators of gene expression and are deregulated in cancer. To characterize hereditary breast tumors based on their miRNA expression profiles we performed global microarray miRNA expression profiling on a retrospective cohort of 80 FFPE breast tissues, including 66 hereditary breast tumors (13 BRCA1, 10 BRCA2 and 43 BRCAX), 10 sporadic breast carcinomas and 4 normal breast tissues, using Exiqon miRCURY LNA™ microRNA Array v.11.0. Here we describe in detail the miRNA microarray expression data and tumor samples used for the study of BRCAX tumor heterogeneity (Tanic et al., 2013) and biomarkers associated with positive BRCA1/2 mutation status (Tanic et al., 2014). Additionally, we provide the R code for data preprocessing and quality control.