Giovanna Cardiero

Background: Early diagnosis of familial hypercholesterolemia (FH) is crucial to improve long-term outcomes. FH diagnosis relies on elevated low-density lipoprotein cholesterol (LDL-C) levels, familial clinical characteristics, and identification of pathogenic variants in FH-related genes. Secondary factors, such as overweight and obesity, are known to influence lipid profiles in the general population. More recently, polygenic risk scores based on single-nucleotide polymorphisms (SNPs) have been proposed as additional determinants of LDL-C levels. Methods: We enrolled 214 pediatric subjects with LDL-C levels ≥95th percentile (after 6 months of dietary intervention) and with at least one parent with LDL-C levels ≥ 95th percentile. All participants underwent biochemical and auxological assessment and genetic testing for FH. In a subgroup of 60 subjects, LDL-C polygenic scores based on 6- and 12-SNPs were calculated. Results: Pathogenic variants confirming heterozygous FH were identified in 190 subjects (variant-positive, V+); 17 were variant-negative (V−), yielding a mutation detection rate of 91.8%. An additional seven patients carrying variants of uncertain significance were excluded from the primary analysis. LDL-C was modestly higher in V+ than V− subjects using both Friedewald (212 vs. 188 mg/dL; p = 0.035) and Martin–Hopkins formulas (208 vs. 187 mg/dL; p = 0.041), while the other main clinical and laboratory parameters were similar. In V+, LDL-C was higher in subjects with null variants, compared to those with defective variants. Body mass index (BMI SDS) was inversely correlated with HDL-C (p < 0.001), and obesity (BMI z-score > 2 SDS) was associated with lower HDL-C and higher LDL-C, non-HDL-C, and ApoB. With regard to the polygenic scores, 12- and 6-SNP scores showed overlap between V+ and V−, and published cut-offs did not discriminate lipid severity in our population; however, in V+ subjects, the 12-SNP score acted as a phenotype modifier, being independently associated with higher LDL-C and non-HDL-C levels after adjustment for age, sex, and BMI SDS. Conclusions: In children selected by LDL-C ≥ 95th percentile, together with autosomal dominant familial hypercholesterolemia, genetic confirmation of FH is achieved in the vast majority of cases. Variant type (null vs. defective), BMI, and polygenic background contribute to phenotypic heterogeneity, supporting the need to address other factors alongside genetic diagnosis. Further validation is needed before polygenic scores can be implemented in routine clinical practice.

Familial Hypercholesterolemia (FH) is a common genetic disorder characterized by elevated LDL-cholesterol levels and an increased risk of premature cardiovascular disease. While pathogenic variants in LDLR, APOB, and PCSK9 are well-established causes, a substantial proportion of clinically suspected FH cases do not carry either pathogenic variants or rare variants of uncertain significance in these genes (FH/V−/USV−). This study aimed to characterize the metabolome/lipidome of genetically confirmed heterozygous FH (HeFH) patients compared to FH/V−/USV−, seeking to identify specific alterations associated with genetic status and phenotypic variability. Untargeted high-resolution mass spectrometry (UHPLC-Q-Exactive-MS)-based lipidomics and nuclear magnetic resonance-based metabolomics were performed on plasma samples of FH patients (n = 20 HeFH and n = 19 FH/V−/USV−) towards healthy controls (n = 22). PLS-DA analysis revealed group-level separation, suggesting differences in the circulating metabolome/lipidome. As expected, most of identified lipid classes were higher in both FH groups compared to normolipidemic controls. Notably, significant lipids (VIP > 1, p < 0.05) showed potential in distinguishing HeFH and FH/V−/USV− patients, particularly sphingomyelins. These data were confirmed by multivariable regression analysis controlling for age, sex, and lipid-lowering therapy as well as by ROC analysis. The evidence of a distinct lipidome signature in the HeFH subgroup may relate to the increased cardiovascular risk of HeFH patients compared to patients without pathogenic variants.

Background: The antisense oligonucleotide against APOC3 mRNA volanesorsen was recently introduced to treat Familial Chylomicronemia Syndrome (FCS). Cases of decreased platelet count are reported among patients treated with volanesorsen. The aim of the study was to evaluate platelet function and thrombin generation (TG) assessment in FCS patients receiving volanesorsen. We performed a cross-sectional study on FCS patients treated with volanesorsen. Methods: Changes in platelet count PLC were assessed from baseline to Tw12 and Tw36. To assess TG, samples were processed by CAT (with PPP-reagent LOW). The results were expressed by the thrombogram graphic (thrombin variation over time); LagTime; endogenous thrombin potential (ETP); peak; time to reach peak (ttpeak), StartTail and Velocity Index. Platelet aggregation was assessed by testing different agonists using the turbidimetry method. Results: Four FCS patients and four matched healthy controls were included in the present study. Changes in PLC were 30% at Tw12 and 34% at Tw36. Thrombin generation results showed values in the normal range (for patients and controls, respectively, LagTime:10.42 ± 4.40 and 9.25 ± 0.99; ttPeak:14.33 ± 4.01 and 13.10 ± 0.67; StartTail: 32.13 ± 3.54 and 29.46 ± 1.69; Velocity Index: 20.21 ± 3.63 and 33.05 ± 13.21; ETP: 599.80 ± 73.47 and 900.2 ± 210.99; peak value: 76.84 ± 1.07 and 123.30 ± 39.45) and no significant difference between cases and controls. Platelet aggregation test showed values in range, with no significant difference compared to healthy controls. Conclusions: Our study showed for the first time that no significant changes in general hemostasis assessed by TG and in platelet function were observed in FCS patients receiving volanesorsen.

Background. Alagille syndrome (ALGS) is a rare autosomal dominant genetic disease caused by pathogenic variants in two genes: Jagged Canonical Notch Ligand 1 (JAG1) and Notch Receptor 2 (NOTCH2). It is characterized by phenotypic variability and incomplete penetrance with multiorgan clinical signs. Methods. Using Next Generation Sequencing (NGS), we analyzed a panel of liver-disease-related genes in a population of 230 patients with cholestasis and hepatopathies. For the rare variants, bioinformatics predictions and pathogenicity classification were performed. Results. We identified eleven rare NOTCH2 variants in 10 patients, two variants being present in the same patient. Ten variants had never been described before in the literature. It was possible to classify only two null variants as pathogenic, whereas the most of variants were missense (8 out of 11) and were classified as uncertain significance variants (USVs). Among patients with ALGS suspicion, two carried null variants, two carried variants predicted to be pathogenic by bioinformatics, one carried a synonymous variant and variants in glycosylation-related genes, and two carried variants predicted as benign in the PEST domain. Conclusions. Our results increased the knowledge about NOTCH2 variants and the related phenotype, allowing us to improve the genetic diagnosis of ALGS.

Background: The antisense oligonucleotide against APOC3 mRNA volanesorsen was recently introduced to treat Familial Chylomicronemia Syndrome (FCS). Cases of decreased platelet count are reported among patients treated with volanesorsen. The aim of the study is to evaluate platelet function and thrombin generation (TG) assessment in FCS patients receiving volanesorsen. We performed a cross-sectional study on FCS patients treated with volanesorsen. Methods: changes in PLC were assessed from baseline to Tw12 and Tw36. To assess TG, samples were processed by CAT (with PPP-reagent LOW). The results were expressed by the trombogram graphic (thrombin variation over time); lagTime; Endogenous Thormbin Potential(ETP); peak; time to reach peak(ttpeak), startTail e velocity Index. Platelet aggregation was assessed by testing different agonists using turbidimetry method. Results: Four FCS patients and 4 matched healthy controls were included in the present study. Changes in PLC at Tw12 was 30% and 34% at T36w. Thrombin generation results show values in normal range (for patients and controls respectively lagTime:10,42±4,40 and 9,25±0,99; ttPeak:14,33± 4,01 and 13,10±0,67; startTail:32,13±3,54 and 29,46±1,69; velocityIndex:20,21±3,63 and 33,05±13,21; ETP 599,80±73,47 and 900,2±210,99; peak value 76,84±1,07 and 123,30±39,45) and no significant difference between cases and controls. Platelet aggregation test showed values in range with no significant difference with healthy controls Conclusions: Our study showed for the first time that no significant changes in the general haemostasis assessed by TG and in platelet function have been observed in FCS patients receiving volanesorsen.

Background: The diagnosis of familial hypercholesterolemia (FH) in children is primarily based on main criteria including low-density lipoprotein cholesterol (LDL-C) levels, increased in the proband and relatives, and its inheritance. Two other relevant parameters are symptoms, rarely occurring in children, as rare are the FH homozygous patients, and the mutation detection of related genes. The latter allows the final diagnosis, although it is not commonly available. Moreover, the application of diagnostic scores, useful in adults, is poorly applied in children. The aim of this study was to compare the reliability of criteria here applied with different scores, apart from genetic analysis, for FH diagnosis. The latter was then confirmed by genetic analysis. Methods: n. 180 hypercholesterolemic children (age 10.2 ± 4.6 years) showing LDL-C levels ≥95th percentile (age- and sex-related), the dominant inheritance pattern of hypercholesterolemia (including LDL-C ≥95th percentile in one parent), were considered potentially affected by FH and included in the study. The molecular analysis of the LDLR, APOB and PCSK9 genes was applied to verify the diagnostic accuracy. Biochemical and family history data were also retrospectively categorized according to European Atherosclerosis Society (EAS), Simon Broome Register (SBR), Pediatric group of the Italian LIPIGEN (LIPIGEN-FH-PED) and Dutch Lipid Clinic Network (DLCN) criteria. Detailed kindred biochemical and clinical assessments were extended to three generations. The lipid profile was detected by standard laboratory kits, and gene analysis was performed by traditional sequencing or Next-Generation Sequencing (NGS). Results: Among 180 hypercholesterolemic subjects, FH suspected based on the above criteria, 164/180 had the diagnosis confirmed, showing causative mutations. The mutation detection rate (MDR) was 91.1%. The scoring criteria proposed by the EAS, SBR and LIPIGEN-FH-PED (resulting in high probable, possible-defined and probable-defined, respectively) showed high sensitivity (~90%), low specificity (~6%) and high MDR (~91%). It is noteworthy that their application, as a discriminant for the execution of the molecular investigation, would lead to a loss of 9.1%, 9.8% and 9.1%, respectively, of FH-affected patients, as confirmed by the genetic analysis. DLCN criteria, for which LDL-C cut-offs are not specific for childhood, would lead to a loss of 53% of patients with mutations. Conclusions: In the pediatric population, the combination of LDL-C ≥95th percentile in the proband and the dominant inheritance pattern of hypercholesterolemia, with LDL-C ≥95th percentile in one parent, is a simple, useful and effective diagnostic criterion, showing high MDR. This pattern is crucial for early FH diagnosis. EAS, SBR and LIPIGEN-FH-PED criteria can underestimate the real number of patients with gene mutations and cannot be considered strictly discriminant for the execution of molecular analysis.

Background: Familial hypercholesterolemia (FH) comprises high LDL-cholesterol (LDL-c) levels and high cardiovascular disease risk. In the absence of pathogenic variants in causative genes, a polygenic basis was hypothesized. Methods: In a population of 418 patients (excluding homozygotes) with clinical suspicion of FH, the FH-causative genes and the regions of single nucleotide polymorphisms (SNPs) included in 12-SNP and 6-SNP scores were sequenced by next-generation sequencing, allowing for the detection of pathogenic variants (V+) in 220 patients. To make a comparison, only patients without uncertain significance variants (V−/USV−) were considered (n = 162). Results: Higher values of both scores were observed in V+ than in V−. Considering a cut-off leading to 80% of V−/USV− as score-positive, a lower prevalence of patients positive for both 12-SNP and 6-SNP scores was observed in V+ (p = 0.010 and 0.033, respectively). Mainly for the 12-SNP score, among V+ patients, higher LDL-c levels were observed in score-positive (223 mg/dL -IQR 187-279) than in negative patients (212 mg/dL -IQR 162–240; p = 0.006). Multivariate analysis confirmed the association of scores and LDL-c levels independently of age, sex, and presence of pathogenic variants and revealed a greater association in children. Conclusions: The 12-SNP and 6-SNP polygenic scores could explain hypercholesterolemia in patients without pathogenic variants as well as the variability of LDL-c levels among patients with FH-causative variants.

Inflammation plays a crucial role in worsening coronavirus disease (COVID-19). Calprotectin is a pro-inflammatory molecule produced by monocytes and neutrophilic granulocytes. The aim of the study was to evaluate both the prognostic role of circulating calprotectin levels and neutrophil count toward fatal outcome in COVID-19 patients. We retrospectively collected and analyzed data on 195 COVID-19 adult patients, 156 hospitalized in the infectious disease unit and 39 in the intensive care unit (ICU). Calprotectin levels and neutrophil counts measured at the first hospitalization day were higher in the patients with a fatal outcome than in surviving ones. The association of high calprotectin levels and neutrophil count to patient death remain significant by logistic regression, independent of patient age. ROC curves analysis for calprotectin levels and neutrophil count revealed a good discriminatory power toward survival (area under the curve of 0.759 and 0.843, respectively) and identified the best cut-off (1.66 mg/L and 16.39 × 10³/µL, respectively). Kaplan–Meier analysis confirmed the prognostic role of high calprotectin levels and neutrophil count in death prediction. In conclusion, this study highlights that calprotectin levels together with neutrophil count should be considered as biomarkers of mortality in COVID-19 patients.