the study of familial hypercholesterolemia in italy a narrative review

Abstract In this review we outline our experience in the clinical and molecular diagnosis of familial hypercholesterolemia (FH), built up over more than three decades. We started our work by selecting FH patients on the basis of stringent clinical criteria, including extensive family studies. In most patients we confirmed the clinical diagnosis by showing a reduced LDLR activity in skin fibroblasts. After the isolation of LDLR cDNA and the characterization of the corresponding gene by the Dallas group, we started a systematic molecular investigation of our patients first using Southern blotting, and, subsequently Sanger sequencing. Up to now we have been able to identify 260 mutations of LDLR gene in more than 1000 genotyped FH patients, including 68 homozygotes. During this survey we identified 13 mutation clusters located in different geographical districts, which gave us the chance to compare the phenotype of patients carrying the most common mutations. We also found that mutations in APOB and PCSK9 genes were a rare cause of FH in our cohort. Despite our efforts, we failed to identify mutations in candidate genes in ∼20% of cases of definite FH. An exome-wide study, conducted within the context of an international collaboration, excluded the presence of other major genes in our unexplained FH cases. Recently, we have adopted sequencing technology of the next generation (NGS) with the parallel sequencing of a panel of FH targeted genes as a way of obtaining a more comprehensive picture of the gene variants potentially involved in the disease

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis

Background: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. Methods: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. Results: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. Conclusions: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour

The role of endocannabinoids in gonadal function and fertility along the evolutionary axis.

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells

Effect of Plasma Unit Weight and Donor Sex on Post-Donation Citrate Level: An Experimental Study on Plasmapheresis Donors

Introduction: Plasmapheresis donation is considered safe and well tolerated, although long-term effects need to be clarified. The volumes of anticoagulant (ACD-A) used are variable and depend primarily on hematocrit (HCT), total blood processed, amount of plasma collected, and donor characteristics. To elucidate the effect of the plasma unit weight setting on plasmapheresis efficiency and ACD-A distribution, we enrolled male donors undergoing a controlled apheresis process donating 700 g and 720 g in two different sessions. In parallel, we investigated a possible effect of sex, recruiting women donating 700 g of plasma. Methods: The study was conducted on men donating 720 g and (12 months later) 700 g of plasma, and on women donating 700 g of plasma. The main outcomes were pre-/post-donation delta (Delta) citrate concentration in donor plasma and ACD-A reinfused to the donor. Information concerning the annual check-up and the procedure was also collected. Intergroup comparisons (men donating 720 g vs. men donating 700 g and men vs. women both donating 700 g) and intragroup associations with donor and procedural characteristics were reported. Results: With the procedure set at 720 g, the machine processed around 44 mL more whole blood to collect 20 g more plasma, and 720 g donors received around 12 mL more anticoagulant than 700 g donors. Accordingly, Delta citrate concentration was 1.5 times higher (12 mu m), with a greater variability observed for 720 g donations. Citrate concentration in the plasma unit was lower in the 720 g group, although not significantly. Comparing outcomes between women and men donating 700 g, we observed higher (and highly variable) Delta citrate and reinfused ACD-A in women, accompanied by lower anticoagulant levels in the unit. Increased Delta citrate is inversely associated with HCT and age in men and with HCT and triglycerides in women. Reinfused ACD-A correlates with HCT in women but not in men. Conclusion: Unit weight setting and sex influence an ACD-A shift from the estimated values toward an increased reinfusion to donor. In parallel, we observed an impact of age and sex on post-donation citrate metabolism. Altogether, these elements should be taken into account for the development of tailored approaches aimed at maintaining similar safety profiles for all donors using different plasmapheresis settings

Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject

Familial hypercholesterolaemia (FH) is an autosomal dominant dyslipidaemia, characterised by elevated LDL cholesterol (LDL-C) levels in the blood. Three main genes are involved in FH diagnosis: LDL receptor (LDLr), Apolipoprotein B (APOB) and Protein convertase subtilisin/kexin type 9 (PCSK9) with genetic mutations that led to reduced plasma LDL-C clearance. To date, several PCSK9 gain-of-function (GOF) variants causing FH have been described based on their increased ability to degrade LDLr. On the other hand, mutations that reduce the activity of PCSK9 on LDLr degradation have been described as loss-of-function (LOF) variants. It is therefore important to functionally characterise PCSK9 variants in order to support the genetic diagnosis of FH. The aim of this work is to functionally characterise the p.(Arg160Gln) PCSK9 variant found in a subject suspected to have FH. Different techniques have been combined to determine efficiency of the autocatalytic cleavage, protein expression, effect of the variant on LDLr activity and affinity of the PCSK9 variant for the LDLr. Expression and processing of the p.(Arg160Gln) variant had a result similar to that of WT PCSK9. The effect of p.(Arg160Gln) PCSK9 on LDLr activity is lower than WT PCSK9, with higher values of LDL internalisation (13%) and p.(Arg160Gln) PCSK9 affinity for the LDLr is lower than WT, EC50 8.6 ± 0.8 and 25.9 ± 0.7, respectively. The p.(Arg160Gln) PCSK9 variant is a LOF PCSK9 whose loss of activity is caused by a displacement of the PCSK9 P’ helix, which reduces the stability of the LDLr-PCSK9 complex.This research was funded by Grupos Consolidados Gobierno Vasco 2021, grant number IT1720-22. A.L.-S. was supported by a grant PIF (2019–2020), Gobierno Vasco and partially supported by Fundación Biofísica Bizkaia. S.J-B. was supported by a Margarita Salas Grant 2022 from the University of the Basque Country

Head-to-head comparison of plasma cTnI concentration values measured with three high-sensitivity methods in a large Italian population of healthy volunteers and patients admitted to emergency department with acute coronary syndrome: A multi-center study

Abstract Background The study aim is to compare cTnI values measured with three high-sensitivity (hs) methods in apparently healthy volunteers and patients admitted to emergency department (ED) with acute coronary syndrome enrolled in a large multicentre study. Methods Heparinized plasma samples were collected from 1511 apparently healthy subjects from 8 Italian clinical institutions (mean age: 51.5 years, SD: 14.1 years, range: 18–65 years, F/M ratio:0.95). All volunteers denied chronic or acute diseases and had normal values of routine laboratory tests. Moreover, 1322 heparinized plasma sample were also collected by 9 Italian clinical institutions from patients admitted to ED with clinical symptoms typical of acute coronary syndrome. The reference study laboratory assayed all plasma samples with three hs-methods: Architect hs-cTnI, Access hs-cTnI and ADVIA Centaur XPT methods. Principal Component Analysis (PCA) was also used to analyze the between-method differences among hs-cTnI assays. Results On average, a between-method difference of 31.2% CV was found among the results of hs-cTnI immunoassays. ADVIA Centaur XPT method measured higher cTnI values than Architect and Access methods. Moreover, 99th percentile URL values depended not only on age and sex of reference population, but also on the statistical approach used for calculation (robust non-parametric vs bootstrap). Conclusions Due to differences in concentrations and reference values, clinicians should be advised that plasma samples of the same patient should be measured for cTnI assay in the same laboratory. Specific clinical studies are needed to establish the most appropriate statistical approach to calculate the 99th percentile URL values for hs-cTnI methods

The SL2S Galaxy-scale Lens Sample. II. Cosmic evolution of dark and luminous mass in early-type galaxies

We present a joint gravitational lensing and stellar-dynamical analysis of 11 early-type galaxies (median deflector redshift \zd=0.5) from Strong Lenses in the Legacy Survey (SL2S). Using newly measured redshifts and stellar velocity dispersions from Keck spectroscopy with lens models from Paper I, we derive the total mass density slope inside the Einstein radius for each of the 11 lenses. The average total density slope is found to be $= 2.16^{+0.09}_{-0.09}$ ($\rho_{\rm tot}\propto r^{-\gamma'}$), with an intrinsic scatter of $0.25^{+0.10}_{-0.07}$. We also determine the dark matter fraction for each lens within half the effective radius, and find the average projected dark matter mass fraction to be $0.42^{+0.08}_{-0.08}$ with a scatter of $0.20^{+0.09}_{-0.07}$ for a Salpeter IMF. By combining the SL2S results with those from the Sloan Lens ACS Survey (median \zd=0.2) and the Lenses Structure and Dynamics survey (median \zd=0.8), we investigate cosmic evolution of $\gamma'$ and find a mild trend \partial/\partial\zd = -0.25^{+0.10}_{-0.12}. This suggests that the total density profile of massive galaxies has become slightly steeper over cosmic time. If this result is confirmed by larger samples, it would indicate that dissipative processes played some role in the growth of massive galaxies since $z\sim1$.Comment: 21 pages, 16 figures, submitted to Ap

Modifications of Chest CT Body Composition Parameters at Three and Six Months after Severe COVID-19 Pneumonia: A Retrospective Cohort Study

We aimed to describe body composition changes up to 6-7 months after severe COVID-19 and to evaluate their association with COVID-19 inflammatory burden, described by the integral of the C-reactive protein (CRP) curve. The pectoral muscle area (PMA) and density (PMD), liver-to-spleen (L/S) ratio, and total, visceral, and intermuscular adipose tissue areas (TAT, VAT, and IMAT) were measured at baseline (T0), 2-3 months (T1), and 6-7 months (T2) follow-up CT scans of severe COVID-19 pneumonia survivors. Among the 208 included patients (mean age 65.6 ± 11 years, 31.3% females), decreases in PMA [mean (95%CI) -1.11 (-1.72; -0.51) cm2] and in body fat areas were observed [-3.13 (-10.79; +4.52) cm2 for TAT], larger from T0 to T1 than from T1 to T2. PMD increased only from T1 to T2 [+3.07 (+2.08; +4.06) HU]. Mean decreases were more evident for VAT [-3.55 (-4.94; -2.17) cm2] and steatosis [L/S ratio increase +0.17 (+0.13; +0.20)] than for TAT. In multivariable models adjusted by age, sex, and baseline TAT, increasing the CRP interval was associated with greater PMA reductions, smaller PMD increases, and greater VAT and steatosis decreases, but it was not associated with TAT decreases. In conclusion, muscle loss and fat loss (more apparent in visceral compartments) continue until 6-7 months after COVID-19. The inflammatory burden is associated with skeletal muscle loss and visceral/liver fat loss

Evaluation of 99th percentile and reference change values of a high-sensitivity cTnI method: A multicenter study

Abstract Background The Italian Society of Clinical Biochemistry (SIBioC) and the Italian Section of the European Ligand Assay Society (ELAS) have recently promoted a multicenter study (Italian hs-cTnI Study) with the aim to accurately evaluate analytical performances and reference values of the most popular cTnI methods commercially available in Italy. The aim of this article is to report the results of the Italian hs-cTnI Study concerning the evaluation of the 99th percentile URL and reference change (RCV) values around the 99th URL of the Access cTnI method. Materials and methods Heparinized plasma samples were collected from 1306 healthy adult volunteers by 8 Italian clinical centers. Every center collected from 50 to 150 plasma samples from healthy adult subjects. All volunteers denied the presence of chronic or acute diseases and had normal values of routine laboratory tests (including creatinine, electrolytes, glucose and blood counts). An older cohort of 457 adult subjects (mean age 63.0 years; SD 8.1 years, minimum 47 years, maximum 86 years) underwent also ECG and cardiac imaging analysis in order to exclude the presence of asymptomatic cardiac disease. Results and conclusions The results of the present study confirm that the Access hsTnI method using the DxI platform satisfies the two criteria required by international guidelines for high-sensitivity methods for cTn assay. Furthermore, the results of this study confirm that the calculation of the 99th percentile URL values are greatly affected not only by age and sex of the reference population, but also by the statistical approach used for calculation of cTnI distribution parameters

Performance evaluation of the high sensitive troponin I assay on the Atellica IM analyser

The Fourth Universal Definition of Myocardial Infarction Global Taskforce recommends the use of high sensitive troponin (hs-Tn) assays in the diagnosis of acute myocardial infarction. We evaluated the analytical performance of the Atellica IM High-sensitivity Troponin I Assay (hs-TnI) (Siemens Healthcare Diagnostics Inc., Tarrytown, USA) and compared its performance to other hs-TnI assays (Siemens Advia Centaur, Dimension Vista, Dimension EXL, and Abbott Architect (Wiesbaden, Germany)) at one or more sites across Europe. Precision, detection limit, linearity, method comparison, and interference studies were performed according to Clinical and Laboratory Standards Institute protocols. Values in 40 healthy individuals were compared to the manufacturer’s cut-offs. Sample turnaround time (TAT) was examined. Imprecision repeatability CVs were 1.1–4.7% and within-lab imprecision were 1.8–7.6% (10.0–25,000 ng/L). The limit of blank (LoB), detection (LoD), and quantitation (LoQ) aligned with the manufacturer’s values of 0.5 ng/L, 1.6 ng/L, and 2.5 ng/L, respectively. Passing-Bablok regression demonstrated good correlations between Atellica IM analyser with other systems; some minor deviations were observed. All results in healthy volunteers fell below the 99th percentile URL, and greater than 50% of each sex demonstrated values above the LoD. No interference was observed for biotin (≤ 1500 µg/L), but a slight bias at 5.0 g/L haemoglobin and 50 ng/L Tn was observed. TAT from was fast (mean time = 10.9 minutes) and reproducible (6%CV). Real-world analytical and TAT performance of the hs-TnI assay on the Atellica IM analyser make this assay fit for routine use in clinical laboratories