Francesca Fayer

Prevalence of ANGPTL3 and APOB Gene Mutations in Subjects With Combined Hypolipidemia

Objective—Mutations of the ANGPTL3 gene have been associated with a novel form of primary hypobetalipoproteinemia, the combined hypolipidemia (cHLP), characterized by low total cholesterol and low HDL-cholesterol levels. The aim of this work is to define the role of ANGPTL3 gene as determinant of the combined hypolipidemia phenotype in 2 large cohorts of 913 among American and Italian subjects with primary hypobetalipoproteinemia (total cholesterol <5th percentile). Methods and Results—The combined hypolipidemia cut-offs were chosen according to total cholesterol and HDL-cholesterol levels reported in the ANGPTL3 kindred described to date: total cholesterol levels, <2nd percentile and HDL-cholesterol, levels <2nd decile. Seventy-eight subjects with combined hypolipidemia were analyzed for ANGPTL3 and APOB genes. We identified nonsense and/or missense mutations in ANGPTL3 gene in 8 subjects; no mutations of the APOB gene were found. Mutated ANGPTL3 homozygous/compound heterozygous subjects showed a more severe biochemical phenotype compared to heterozygous or ANGPTL3 negative subjects, although ANGPTL3 heterozygotes did not differ from ANGPTL3 negative subjects. Conclusion—These results demonstrated that in a cohort of subjects with severe primary hypobetalipoproteinemia the prevalence of ANGPTL3 gene mutations responsible for a combined hypolipidemia phenotype is about 10%, whereas mutations of APOB gene are absent.

FragClust and TestClust, two informatics tools for chemical structure hierarchical clustering analysis applied to lipidomics. The example of Alzheimer's disease.

AbstractLipidomic analysis is able to measure simultaneously thousands of compounds belonging to a few lipid classes. In each lipid class, compounds differ only by the acyl radical, ranging between C10:0 (capric acid) and C24:0 (lignoceric acid). Although some metabolites have a peculiar pathological role, more often compounds belonging to a single lipid class exert the same biological effect. Here, we present a lipidomics workflow that extracts the tandem mass spectrometry data from individual files and uses them to group compounds into structurally homogeneous clusters by chemical structure hierarchical clustering analysis (CHCA). The case-to-control peak area ratios of the metabolites are then analyzed within clusters. We created two freely available applications to assist the workflow: FragClust to generate the tables to be subjected to CHCA, and TestClust to perform statistical analysis on clustered data. We used the lipidomics data from the plasma of Alzheimers disease (AD) patients in comparison with healthy controls to test the workflow. To date, the search for plasma biomarkers in AD has not provided reliable results. This article shows that the workflow is helpful to understand the behavior of whole lipid classes in plasma of AD patients. Graphical AbstractChemical Hierarchical Cluster Analysis applied to Lipidomics. Software assisted workflow.

Searching for wheat plants with low toxicity in celiac disease: Between direct toxicity and immunologic activation.

BACKGROUND Natural or induced variations in the noxiousness of gluten proteins for celiac disease (CD) patients are currently being investigated for their potential in breeding wheat crops with reduced toxicity. AIMS We evaluated the bread wheat line C173 for its effects on the in vitro-grown duodenal mucosa of CD patients. METHODS In vitro-grown duodenal mucosa biopsies of 19 CD patients on a gluten-free diet were exposed to peptic/tryptic-digested prolamins from bread wheat line C173 lacking gliadin-glutenin subunits, analyzed for morphology, cytokine and anti-tTG antibody production, and compared with mucosa biopsies exposed to prolamins from wild-type cv. San Pastore. RESULTS Duodenal mucosa biopsies exposed to prolamins from C173 and San Pastore released higher amounts of IFN-γ, IL-2, IL-10 and anti-tTG antibodies in the culture medium than untreated controls. The line C173 differed from cv. San Pastore as it did not produce negative effects on enterocyte height, suggesting that manipulating prolamin composition can affect innate immune responses of CD mucosa to wheat gluten. CONCLUSIONS Our data demonstrated that this gliadin-deficient wheat has a lower direct toxicity but activates an immunologic reaction of the duodenal mucosa like that of the common wheat species.

Obesity and the metabolic syndrome in a student cohort from Southern Italy

BACKGROUND AND AIM Cardiovascular (CV) risk factors present in childhood predict future CV events. Few data regarding the metabolic syndrome (MS) prevalence are available in adolescents from Mediterranean areas where obesity is becoming a social emergency. This study presents data of MS prevalence in a student cohort from southern Italy. METHODS AND RESULTS 1629 students between 7 and 14 years of age underwent anthropometric measurements and a blood sample was obtained to assess biochemical parameters. MS risk factors were calculated based on age and gender adjusted percentiles of parameter distributions. MS prevalence rate was 0.022 using paediatric, age-adjusted criteria; the rate increased to 0.029 using a 90th percentile criteria for fasting blood glucose instead of >100mg/dL. Using the criteria issued by the International Diabetes Federation the MS prevalence rate dropped to 0.005. The exploratory factor analysis identified four factors: age/fat related, lipids, blood pressure and blood glucose. Family history of type 2 diabetes mellitus was associated with triglyceride [OR=1.55 (1.0-2.3)] and BMI [OR=1.71 (1.2-2.4)] but not to blood glucose by logistic regression analysis. CONCLUSIONS In a student cohort from Southern Italy, obesity is associated with the features of MS.

Familial hypobetalipoproteinemia due to apolipoprotein B R463W mutation causes intestinal fat accumulation and low postprandial lipemia

OBJECTIVE Familial hypobetalipoproteinemia (FHBL) is characterized by inherited low plasma levels of apolipoprotein B (apoB)-containing lipoproteins. In this paper we investigated whether the already described APOB R463W missense mutation, a FHBL mutation able to impair the activity of microsomal triglyceride transfer protein (MTP), may cause intestinal fat accumulation and reduced postprandial lipemia. METHODS Four out of five probands harboring APOB R463W mutation were compared with six healthy controls and six patients with celiac disease (CD). An oral fat load supplemented with retinyl palmitate (RP) was administered and a gastro-duodenal endoscopy with biopsy was performed. RESULTS Plasma triglyceride area under curves was significantly reduced in FHBL probands compared to controls and CD patients; the proportion of absorbed RP was similar to that of CD patients. Only the intestinal biopsies of FHBL patients showed lipids accumulating within the duodenal mucosa. CONCLUSIONS FHBL due to R463W apoB mutation is a cause of intestinal fat accumulation and postprandial lipid absorption impairment.

Plasma Non―cholesterol Sterols: A Useful Diagnostic Tool in Pediatric Hypercholesterolemia

Current guidelines strongly recommend the identification of genetic forms of hypercholesterolemia (HC) during childhood. The usefulness of non–cholesterol sterols (NCS) in the diagnosis of genetic HC has not been fully explored. Plasma NCS were measured by gas chromatography/mass spectrometry (GC/MS) in 113 children with hypercholesterolemia affected by: autosomal dominant hypercholesterolemia (ADH), familial combined hyperlipidemia (FCHL), polygenic hypercholesterolemia (PHC), and in 79 controls to evaluate: i) plasma NCS profile in different genetic HC and ii) the usefulness of NCS for the diagnosis of HC beyond current clinical criteria. ADH was characterized by raised lathosterol/total cholesterol (TC) and reduced phytosterols/TC ratios, indicative of increased cholesterol synthesis. FCHL showed a slight increase of lathosterol/TC ratio, whereas PHC showed increased phytosterols/TC ratios, indicative of increased cholesterol absorption. In a post hoc discriminant analysis of patients with HC, lipid values correctly classified the 73% (14 of 19) of ADH, whereas the inclusion of plasma sterols allowed the correct identification of all 19 patients with ADH. FCHL was not differentiated from PHC (62 versus 69%). In conclusion, NCS measurement showed that cholesterol plasma levels are related to the cholesterol synthesis in ADH and to cholesterol absorption in PHC. NCS improve the detection of ADH in pediatric patients, whereas FCHL diagnosis is not improved.

Clinical symptoms in celiac patients on a gluten-free diet

Objective. Persistent villous atrophy in patients with celiac disease (CD) on a gluten-free diet (GFD) is reported with increasing frequency. The aim of this study was to evaluate a possible association between persistent damage of the villi and “atypical” gastrointestinal symptoms in CD patients on a GFD. Material and methods. Sixty-nine CD patients on a GFD were divided into two groups: Group A included 42 patients (6 M, 36 F, age range 17–62 years) undergoing esophagogastroduodenoscopies (EGDs) due to the presence of symptoms; Group B included 27 control patients (6 M, 21 F, age range 24–71 years) who were asymptomatic at the time of the study. Both groups underwent EGDs and a duodenal histologic study. Results. Persistent endoscopic lesions were more frequent in Group A (30/42) than in Group B (12/27; p=0.01). Villous atrophy was significantly more frequent in Group A than in Group B: 85% versus 33% (p<0.0001; odds ratio (OR)=12; 95% CI 3.7–38.9). Gastrointestinal symptoms in the Group A patients were different from those present at CD diagnosis: anemia/diarrhea/weight loss in 6 cases; gastroesophageal reflux disease (GERD)-like symptoms in 12 cases; abdominal pain/constipation in 24 cases. In Group A there was no difference in gender distribution, age and duration of GFD between subjects with normal villi and those with persistent partial villous atrophy. Patients with persistent symptoms showed a higher intraepithelial eosinophil count (p=0.005) than the asymptomatic patients (p=0.01). Conclusions. Persistent intestinal villous atrophy in CD patients on a GFD is associated with gastrointestinal symptoms considered “atypical” for CD and not present at CD diagnosis.

Interleukin 6 plasma levels predict with high sensitivity and specificity coronary stenosis detected by coronary angiography

In recent years new biomarkers able to measure the coronary atherosclerotic burden have been investigated. The aim of the present study was: i) to measure plasma levels of four biomarkers: C reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), interleukin 6 (IL-6), 8-isosprostane (8-ISO), in a series of patients undergoing coronary angiography; ii) to assess the power of the biomarkers to predict critical coronary stenosis detected by angiography. The study population consisted of a group of 438 subjects undergoing coronary angiography; 160 patients with 0, 1, 2, or 3 critical vessels were selected, and biomarkers plasma levels were measured in plasma samples obtained before the procedure. The most predictive biomarker was then assayed in 120 patients with critical stenosis and 120 unmatched patients without stenosis. CRP, sICAM-1, IL-6 and 8-ISO plasma levels increased with the number of diseased vessels. All biomarkers were good predictors of critical stenosis (receiver-operator-curve [ROC] areas; CRP = 0.880, IL-6 = 0.936, sICAM-1 = 0.907, 8-ISO = 0.873). IL-6 was confirmed in an expanded sample of 240 subjects to be the best predictor with a ROC area = 0.959. With a threshold of 3.6 ng/l, a 100% sensitivity (120/120) and a 90% specificity (108/120) was observed. In conclusion, IL-6, sICAM-1, CRP and 8-ISO are predictive of CAD. IL-6 predicts critical coronary stenosis with the highest sensitivity and specificity.

Predominance of type 1 innate lymphoid cells in the rectal mucosa of patients with non-celiac wheat sensitivity: reversal after a wheat-free diet.

OBJECTIVES:Non-celiac wheat sensitivity (NCWS) is defined as a reaction to ingested wheat after exclusion of celiac disease and wheat allergy. As its pathogenesis is incompletely understood, we evaluated the inflammatory response in the rectal mucosa of patients with well-defined NCWS.METHODS:The prospective study included 22 patients with irritable bowel syndrome (IBS)-like clinical presentation, diagnosed with NCWS by double-blind placebo-controlled challenge. Eight IBS patients not improving on wheat-free diet were used as controls. Two weeks after oral challenge was performed with 80 grams of wheat daily, cells were isolated from rectal biopsies and thoroughly characterized by fluorescence-activated cell sorting analysis for intracellular cytokines and surface markers.RESULTS:Rectal biopsies from wheat-challenged NCWS patients showed that a significant mucosal CD45+ infiltrate consisted of CD3+ and CD3− lymphocytes, with the latter spontaneously producing more interferon (IFN)-γ than IBS controls. About 30% of IFN-γ-producing CD45+ cells were T-bet+, CD56−, NKP44−, and CD117−, defining them as a type-1 innate lymphoid cells (ILC1). IFN-γ-producing ILC1 cells significantly decreased in 10 patients analyzed 2 weeks after they resumed a wheat-free diet.CONCLUSIONS:These data indicate that, in patients with active NCWS, IFN-γ-producing ILC1 cells infiltrate rectal mucosa and support a role for this innate lymphoid cell population in the pathogenesis of NCWS.

Persistence of Nonceliac Wheat Sensitivity, Based on Long-term Follow-up

We investigated how many patients with a diagnosis of nonceliac wheat sensitivity (NCWS) still experienced wheat sensitivity after a median follow-up time of 99 months. We collected data from 200 participants from a previous study of NCWS, performed between July and December 2016 in Italy; 148 of these individuals were still on a strict wheat-free diet. In total, 175 patients (88%) improved (had fewer symptoms) after a diagnosis of NCWS; 145 of 148 patients who adhered strictly to a gluten-free diet (98%) had reduced symptoms, compared with 30 of 52 patients who did not adhere to a gluten-free diet (58%) (P < .0001). Of the 22 patients who repeated the double-blind, placebo-controlled challenge, 20 reacted to wheat. We conclude that NCWS is a persistent condition. Clinicaltrials.gov registration number: NCT02823522.