Contemporary lipid-lowering management and risk of cardiovascular events in homozygous familial hypercholesterolaemia: insights from the Italian LIPIGEN Registry

Aims: The availability of novel lipid-lowering therapies (LLTs) has remarkably changed the clinical management of homozygous familial hypercholesterolaemia (HoFH). The impact of these advances was evaluated in a cohort of 139 HoFH patients followed in a real-world clinical setting. Methods and results: The clinical characteristics of 139 HoFH patients, along with information about LLTs and low-density lipoprotein cholesterol (LDL-C) levels at baseline and after a median follow-up of 5 years, were retrospectively retrieved from the records of patients enrolled in the LIPid transport disorders Italian GEnetic Network-Familial Hypercholesterolaemia (LIPIGEN-FH) Registry. The annual rates of major atherosclerotic cardiovascular events (MACE-plus) during follow-up were compared before and after baseline. Additionally, the lifelong survival free from MACE-plus was compared with that of the historical LIPIGEN HoFH cohort. At baseline, LDL-C level was 332 ± 138 mg/dL. During follow-up, the potency of LLTs was enhanced and, at the last visit, 15.8% of patients were taking quadruple therapy. Consistently, LDL-C decreased to an average value of 124 mg/dL corresponding to a 58.3% reduction (Pt < 0.001), with the lowest value (∼90 mg/dL) reached in patients receiving proprotein convertase subtilisin/kexin type 9 inhibitors and lomitapide and/or evinacumab as add-on therapies. The average annual MACE-plus rate in the 5-year follow-up was significantly lower than that observed during the 5 years before baseline visit (21.7 vs. 56.5 per 1000 patients/year; P = 0.0016). Conclusion: Our findings indicate that the combination of novel and conventional LLTs significantly improved LDL-C control with a signal of better cardiovascular prognosis in HoFH patients. Overall, these results advocate the use of intensive, multidrug LLTs to effectively manage HoFH

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Principi di Patologia Generale

La Patologia Generale tratta tutti gli aspetti delle malattie, ma dedica particolare attenzione alle cause, ai meccanismi, all'evoluzione ed al significato di quelle condizioni biologiche a-normali, vale a dire rare, che sono le malattie. Premessa indispensabile è infatti comprendere che le interazioni degli agenti patologici con gli organismi producono risposte, generalmente fisiologiche, che solo raramente evolvono in malattia. D’altra parte, paradossalmente, sono talora i circuiti omeostatici a realizzare i deficit funzionali e le lesioni cellulari. Gli effetti tessutali dei processi etiopatogenetici possono essere compresi al microscopio su esempi semplici di patologia cellulare, con un’analisi ragionata meglio se sviluppata individualmente dagli studenti. I meccanismi fisiopatologici possono invece essere illustrati discutendo casi paradigmatici offerti dalla casistica clinica. Le Scienze della Salute sono in così rapido sviluppo che, per chi si accinge ad illustrare i Principi della Patologia Generale, le maggiori difficoltà sono rappresentate dalla non eludibile necessità di selezionare gli argomenti e di decidere il livello d’approfondimento nel trattarli. I criteri di selezione utilizzati sono: 1. la valorizzazione dei risultati delle scienze sperimentali; 2. l'interpretazione dei processi generali di Patologia fino al livello molecolare, senza tuttavia ignorare il valore, non solo didattico, di discutere i quadri di patologia clinica ed anatomica a livello di cellula e tessuto; 3. l'uso privilegiato di criteri anatomo-funzionali nella classificazione ed interpretazione dei processi di Fisiopatologia Generale. Pertanto i capitoli dedicati alla Fisiopatologia Generale non includono una descrizione analitica di tutti gli organi ed apparati. Ad esempio, il sistema ormonale e quelli digestivo ed emuntorio sono discussi nell’ambito di funzioni generali come il controllo idrico e ionico. Abbiamo così voluto dare agli studenti alcune occasioni di ragionamento induttivo e deduttivo del metodo sperimentale, in modo da farli riflettere su principi e problemi di anatomia, biochimica, fisiologia e pertanto patologia. Solo ben dotati di tali strumenti sarà loro possibile, una solida padronanza delle future conoscenze specialistiche. La materia è organizzata in sezioni che superano la proliferazione disciplinare dei curricula accademici italiani. E’ preliminarmente dedicato spazio all'elencazione delle Cause Interne ed Esterne di Patologia (Etiologia) chiarendo di volta in volta i meccanismi (Patogenesi), con cui la lesione prima è indotta ed eventualmente evolve in quadri clinici conclamati. E’ sempre sottolineato il fatto che le malattie sono, salvo poche eccezioni, dei processi patologici complessi. E’ dedicato ampio spazio anche ai meccanismi della loro eventuale guarigione. Prima della trattazione delle Cause Biologiche sono stati introdotti alcuni capitoli di Patologia Cellulare, che permettono di distinguere processi monocausali molecolari da quelli di più complessa natura, quali le neoplasie benigne e maligne. Seguono alcune sezioni dedicate ai meccanismi di risposta alle lesioni (Infiammazione) od anche solo agli agenti chimici o biologici potenzialmente lesivi (Immunologia). Si pone l’accento sin d'ora sul fatto che questi meccanismi di risposta sono essenziali sia nella prevenzione o riparazione di lesioni indotte da cause esterne, sia nell'induzione o nell'amplificazione di lesioni da agenti chimici o biologici, che non sono direttamente lesivi (Immunopatologia). I capitoli classicamente trattati nei manuali di Fisiopatologia Generale sono introdotti da una discussione della nomenclatura complessa e contraddittoria, che ancora domina le specialità mediche e chirurgiche. I successivi capitoli sono quindi organizzati secondo criteri anatomo-funzionali, iniziando con la descrizione delle cause, delle sedi di lesione principale, dei meccanismi di compenso e delle conseguenze funzionali delle anomalie di assorbimento e smaltimento (Fisiopatologia respiratoria) e della distribuzione ed utilizzazione (Fisiopatologia della Circolazione e del Sangue) dell'ossigeno. La fisiopatologia generale del controllo idrico e ionico consente di esemplificare fondamentali circuiti omeostatici di regolazione dell’increzione ormonale e delle funzioni emuntorie. Il manuale si conclude con una sintesi delle innumerevoli funzioni regolatrici del sistema nervoso (Fisiopatologia Generale del Sistema Nervoso) per introdurre gli studenti alla complessità delle attività cerebrali superiori, che fanno di una macchina costituita da 50 miliardi di cellule una Persona. Contiene Atlante per le Esercitazioni di Patologia Generale su C

High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins

Electrophoresis. 1995 Jan;16(1):101-4. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins. Rossini K, Rizzi C, Sandri M, Bruson A, Carraro U. Source Department of Biomedical Sciences, University of Padova, Italy. Abstract In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS-PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also resolved in a complex mixture of isomyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle. PMID: 7737081 [PubMed - indexed for MEDLINE

Purification of Myosin Heavy Chain Isoforms by Electroendosmotic Preparative Gel Electrophoresis: Characterization of Embryonic Slow Myosin Heavy Chain

Myosin is a hexapolypeptide constituted by four light and two heavy chains the isoforms of which segregate differently in specific stages of animal development and in different fiber types in adulthood. In mammals the Myosin Heavy Chains (MHC) are polypeptides with a molecular mass of about 200 KDa which isoforms can be identified by SDS PAGE and/or immunochemistry. A method for the purification of myosin heavy chain isoforms using a SDS Electrendosmotic Preparative Gel Electrophoresis (SDS EPGE) is described. Semplicity and reproducibility of this approach permits purification of single isoforms from complex mixture with a sufficient high recovery to perform immunochemical or biochemical studies. The possibility to apply useful tool as SDS removal and protein concentration by KDS precipitation before further analysis on the sample is described. Application of the methodology to the study of slow type embryonic MHC by enzymatic as well as chemical peptide mapping and amino acid composition is described