Polymorphisms within autophagy-related genes as susceptibility biomarkers for multiple myeloma: a meta-analysis of three large cohorts and functional characterization

Functional data used in this project have been meticulously catalogued and archived in the BBMRI-NL data infrastructure (https://hfgp.bbmri.nl/, accessed on 12 February 2020) using the MOLGENIS open-source platform for scientific data.Multiple myeloma (MM) arises following malignant proliferation of plasma cells in the bone marrow, that secrete high amounts of specific monoclonal immunoglobulins or light chains, resulting in the massive production of unfolded or misfolded proteins. Autophagy can have a dual role in tumorigenesis, by eliminating these abnormal proteins to avoid cancer development, but also ensuring MM cell survival and promoting resistance to treatments. To date no studies have determined the impact of genetic variation in autophagy-related genes on MM risk. We performed meta-analysis of germline genetic data on 234 autophagy-related genes from three independent study populations including 13,387 subjects of European ancestry (6863 MM patients and 6524 controls) and examined correlations of statistically significant single nucleotide polymorphisms (SNPs; p < 1 × 10−9) with immune responses in whole blood, peripheral blood mononuclear cells (PBMCs), and monocyte-derived macrophages (MDM) from a large population of healthy donors from the Human Functional Genomic Project (HFGP). We identified SNPs in six loci, CD46, IKBKE, PARK2, ULK4, ATG5, and CDKN2A associated with MM risk (p = 4.47 × 10−4−5.79 × 10−14). Mechanistically, we found that the ULK4rs6599175 SNP correlated with circulating concentrations of vitamin D3 (p = 4.0 × 10−4), whereas the IKBKErs17433804 SNP correlated with the number of transitional CD24+CD38+ B cells (p = 4.8 × 10−4) and circulating serum concentrations of Monocyte hemoattractant Protein (MCP)-2 (p = 3.6 × 10−4). We also found that the CD46rs1142469 SNP corre lated with numbers of CD19+ B cells, CD19+CD3− B cells, CD5+ IgD− cells, IgM− cells, IgD−IgM− cells, and CD4−CD8− PBMCs (p = 4.9 × 10−4−8.6 × 10−4 ) and circulating concentrations of interleukin (IL)-20 (p = 0.00082). Finally, we observed that the CDKN2Ars2811710 SNP correlated with levels of CD4+EMCD45RO+CD27− cells (p = 9.3 × 10−4 ). These results suggest that genetic variants within these six loci influence MM risk through the modulation of specific subsets of immune cells, as well as vitamin D3−, MCP-2−, and IL20-dependent pathways.This work was supported by the European Union’s Horizon 2020 research and innovation program, N° 856620 and by grants from the Instituto de Salud Carlos III and FEDER (Madrid, Spain; PI17/02256 and PI20/01845), Consejería de Transformación Económica, Industria, Conocimiento y Universidades and FEDER (PY20/01282), from the CRIS foundation against cancer, from the Cancer Network of Excellence (RD12/10 Red de Cáncer), from the Dietmar Hopp Foundation and the German Ministry of Education and Science (BMBF: CLIOMMICS [01ZX1309]), and from National Cancer Institute of the National Institutes of Health under award numbers: R01CA186646, U01CA249955 (EEB).This work was also funded d by Portuguese National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020 and by the project NORTE-01-0145-FEDER-000055, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Commercial products for Artemia enrichment affect growth performance, digestive system maturation, ossification and incidence of skeletal deformities in Senegalese sole (Solea senegalensis) larvae

Proper nutrition at first feeding in marine fish larvae is an important factor for successful larval rearing. However, live prey used to feed marine finfish larvae lack essential fatty acids requiring commercial hatcheries to enrich live prey to provide a balanced diet. Currently, the many commercially available enrichments for live prey present considerable differences in their formulas, and compositions of essential nutrients. Since nutritional requirements are species-specific it is necessary to define those enrichments most appropriate for each species in terms of larval development and quality. This study aimed to evaluate the effect of six enrichments on Senegalese sole larval performance including growth, digestive system development and skeletogenesis, to determine whether any of these products are more suitable for Senegalese sole larval rearing. From 8 to 37 days post hatching (dph), larvae were fed Artemia nauplii previously enriched with six different formulae: Easy Selco© (INVE, ES), Easy Selco© (INVE) half diluted with olive oil (ES/2), Multigain© (BioMar, MG), Red Pepper© (Bernaqua, RP), Aquagrow Gold© (ABN, AGG) and Aquagrow DHA© (ABN, AGD), with the last two being diluted by a third with olive oil. The dietary treatments significantly affected larval growth and performance; larvae fed Artemia enriched with AGG presented significantly higher final dry weight (2.0 ± 0.4 mg), growth and intestinal maturation than larvae fed ES-enriched Artemia (final dry weight: 1.1 ± 0.3 mg). Larvae fed the AGG treatment also were those most advanced in metamorphosis and ossification processes. Larvae fed ES/2, RP, AGD and MG treatments presented intermediary values for all these parameters. No significant effect of the dietary treatments was detected in final survival and incidence of skeletal deformities. We conclude that, among the enrichments tested, AGG is the most appropriate for larvae of Senegalese sole. © 2011.This work was funded by the Ministry of Science and Innovation (MICIIN) and the Spanish National Research Council (CSIC) of the Spanish Government (projects AGL2008-03897-C04-01/ACU, AGL2008-03897-C04-04/ACU and PIF-200930I128). M.J. Darias was supported by a Juan de la Cierva post-doctoral contract.Peer Reviewe

The effect of dietary oxidized lipid levels on growth performance, antioxidant enzyme activities, intestinal lipid deposition and skeletogenesis in Senegalese sole (Solea senegalensis) larvae

Fish tissues, particularly rich in n-3 PUFA, are prone to lipid peroxidation that can damage cellular membranes, cause severe lesions and subsequently incidences of disease and mortality. However, fish possess antioxidant defences, such as vitamin E (VE) and antioxidant enzymes, to protect them against oxidative damage. This study investigated the effects of an increasing gradient of oxidized dietary lipid on the survival, growth performance, skeletogenesis and antioxidant defensive processes occurring in Senegalese sole (Solea senegalensis) larvae. Four groups of fish were fed live prey enriched with experimental emulsions containing an increasing gradient of oxidized oil: non-oxidized, NO+VE, 34.5 nmol MDA g(-1) w.w.; mildly oxidized, MO+VE, 43.1 nmol MDA g(-1) w.w.; highly oxidized, HO+VE, 63.3 nmol MDA g(-1) w.w. and highly oxidized without VE, HO-VE, 78.8 nmol MDA g(-1) w.w. The oxidation levels increased in enriched rotifers following the oxidation gradient of the emulsions, but were not affected in enriched Artemia metanauplii. The oxidation status of Senegalese sole larvae increased during development, but this was not related to the dietary treatments. The increasing dietary oxidation levels did not affect the fatty acid profile, survival, growth performance and metamorphosis processes of sole larvae. Senegalese sole seem to activate antioxidant defence mechanisms in response to the increasing amounts of dietary peroxidized lipids, in a manner efficiently enough to prevent detection of any alterations of these physiological processes. Antioxidant systems and detoxification mechanisms appeared to occur through the consumption of dietary a-tocopherol, the activation of the antioxidant enzymes (catalase, superoxide dismutase, glutathione S-transferase, glutathione reductase) and the retention of oxidized fat in the intestinal enterocytes for detoxification prior to their utilization. However, fish fed the highest oxidized diet presented a reduction in bone mineralization, but lower incidence of deformities in the vertebral and caudal regions than fish fed the other diets. This study exemplifies the importance of rearing Senegalese sole larvae on non-oxidized diets during the early larval development to avoid detrimental consequences in older fish, most notably in the process of skeletogenesis

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukemia

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine or other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In comparison to EBV-negative tumors, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL and exhibited CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal center DLBCL phenotype, EBV latency programs type II/III, and a very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoral B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/-IL2γc-/-mice. Remarkably, the recipients' impaired immunosurveillance favored the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients, but not from healthy donors. Eventually, these cells generated monoclonal tumors (mostly CLL-unrelated but also CLL-related) recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts exhibited indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and BCR signaling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL

Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders

[Introduction]: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype–phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. [Aim]: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. [Methods]: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. [Results]: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. Conclusion: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.Peer Reviewe

Molecular profiling of immunoglobulin heavychain gene rearrangements unveils new potential prognostic markers for multiple myeloma patients

Multiple myeloma is a heterogeneous disease whose pathogenesis has not been completely elucidated. Although B-cell receptors play a crucial role in myeloma pathogenesis, the impact of clonal immunoglobulin heavy-chain features in the outcome has not been extensively explored. Here we present the characterization of complete heavychain gene rearrangements in 413 myeloma patients treated in Spanish trials, including 113 patients characterized by next-generation sequencing. Compared to the normal B-cell repertoire, gene selection was biased in myeloma, with significant overrepresentation of IGHV3, IGHD2 and IGHD3, as well as IGHJ4 gene groups. Hypermutation was high in our patients (median: 8.8%). Interestingly, regarding patients who are not candidates for transplantation, a high hypermutation rate (≥7%) and the use of IGHD2 and IGHD3 groups were associated with improved prognostic features and longer survival rates in the univariate analyses. Multivariate analysis revealed prolonged progression-free survival rates for patients using IGHD2/IGHD3 groups (HR: 0.552, 95% CI: 0.361−0.845, p = 0.006), as well as prolonged overall survival rates for patients with hypermutation ≥7% (HR: 0.291, 95% CI: 0.137−0.618, p = 0.001). Our results provide new insights into the molecular characterization of multiple myeloma, highlighting the need to evaluate some of these clonal rearrangement characteristics as new potential prognostic markers

Molecular profiling of immunoglobulin heavychain gene rearrangements unveils new potential prognostic markers for multiple myeloma patients

Multiple myeloma is a heterogeneous disease whose pathogenesis has not been completely elucidated. Although B-cell receptors play a crucial role in myeloma pathogenesis, the impact of clonal immunoglobulin heavy-chain features in the outcome has not been extensively explored. Here we present the characterization of complete heavychain gene rearrangements in 413 myeloma patients treated in Spanish trials, including 113 patients characterized by next-generation sequencing. Compared to the normal B-cell repertoire, gene selection was biased in myeloma, with significant overrepresentation of IGHV3, IGHD2 and IGHD3, as well as IGHJ4 gene groups. Hypermutation was high in our patients (median: 8.8%). Interestingly, regarding patients who are not candidates for transplantation, a high hypermutation rate (≥7%) and the use of IGHD2 and IGHD3 groups were associated with improved prognostic features and longer survival rates in the univariate analyses. Multivariate analysis revealed prolonged progression-free survival rates for patients using IGHD2/IGHD3 groups (HR: 0.552, 95% CI: 0.361−0.845, p = 0.006), as well as prolonged overall survival rates for patients with hypermutation ≥7% (HR: 0.291, 95% CI: 0.137−0.618, p = 0.001). Our results provide new insights into the molecular characterization of multiple myeloma, highlighting the need to evaluate some of these clonal rearrangement characteristics as new potential prognostic markers