High CD49a+ NK cell infiltrate is associated with poor clinical outcomes in Hepatocellular Carcinoma.

NK cells infiltrating Hepatocellular Carcinoma (HCC) may express residency markers such as Integrin Subunit Alpha 1 (CD49a) that have been associated with nurturing functions in the decidua, and characterized by the production of angiogenic factors as well as loss of cytotoxicity. CIBERSORT, a computational analysis method for quantifying cell fractions from bulk tissue gene expression profiles, was used to estimate the infiltrating immune cell composition of the tumor microenvironment from gene expression profiles of a large cohort of 225 HCCs in the public GEO database. Decidual-like CD49a+ NK cells, in addition to another 22 immune cell populations, were characterized and thoroughly investigated so that HCC cell heterogeneity in a large cohort of 225 HCCs from the public GEO database could be studied. An inverse correlation of the expression of CD49a+ NK-cells and CD8+ T-cells suggested a negative association with clinical outcomes. This result was confirmed in a further validation cohort of 100 HCC patients from The Cancer Genome Atlas, Liver Hepatocellular Carcinoma (TCGA-LIHC). Cox regression analysis did not identify CD49a+ cells as a variable independently associated with survival. However, a more abundant infiltrate of this subset was present in patients at a more advanced pathological and clinical HCC stage. In conclusion, we found that NK cells, with a decidual-like gene expression profile, are enriched in HCC, and their abundance increases not only in tumor size but also at advanced stages of the disease suggesting that these cells play a role in tumor growth. For this reason, these NK cells may represent a possible new target for immunotherapeutic approaches in HCC.

What Is the Current Status of Hepatitis B Virus Viro-Immunology?

The natural history of hepatitis B virus (HBV) infection is closely dependent on the dynamic interplay between the host immune response and viral replication. Spontaneous HBV clearance in acute self-limited infection is the result of an adequate and efficient antiviral immune response. Instead, it is widely recognized that in chronic HBV infection, immunologic dysfunction contributes to viral persistence. Long-lasting exposure to high viral antigens, upregulation of multiple co-inhibitory receptors, dysfunctional intracellular signaling pathways and metabolic alterations, and intrahepatic regulatory mechanisms have been described as features ultimately leading to a hierarchical loss of effector functions up to full T-cell exhaustion.

FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses.

Monitoring antigen-specific T cell frequency and function is essential to assess the host immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we present a FluoroSpot assay for concurrently detecting ex vivo antiviral cytokine production by SARS-CoV-2-specific T cells following peptide stimulation. We then detail intracellular cytokine staining by flow cytometry to further validate the FluoroSpot assay results and define the specific T cell subpopulations. For complete details on the use and execution of this protocol, please refer to Tiezzi et al. (2023).1.

ANGPTL3 Deficiency and Risk of Hepatic Steatosis.

BACKGROUND: ANGPTL3 (angiopoietin-like 3) is a therapeutic target for reducing plasma levels of triglycerides and low-density lipoprotein cholesterol. A recent trial with vupanorsen, an antisense oligonucleotide targeting hepatic production of ANGPTL3, reported a dose-dependent increase in hepatic fat. It is unclear whether this adverse effect is due to an on-target effect of inhibiting hepatic ANGPTL3. METHODS: We recruited participants with ANGPTL3 deficiency related to ANGPTL3 loss-of-function (LoF) mutations, along with wild-type (WT) participants from 2 previously characterized cohorts located in Campodimele, Italy, and St. Louis, MO. Magnetic resonance spectroscopy and magnetic resonance proton density fat fraction were performed to measure hepatic fat fraction and the distribution of extrahepatic fat. To estimate the causal relationship between ANGPTL3 and hepatic fat, we generated a genetic instrument of plasma ANGPTL3 levels as a surrogate for hepatic protein synthesis and performed Mendelian randomization analyses with hepatic fat in the UK Biobank study. RESULTS: We recruited participants with complete (n=6) or partial (n=32) ANGPTL3 deficiency related to ANGPTL3 LoF mutations, as well as WT participants (n=92) without LoF mutations. Participants with ANGPTL3 deficiency exhibited significantly lower total cholesterol (complete deficiency, 78.5 mg/dL; partial deficiency, 172 mg/dL; WT, 188 mg/dL; P<0.05 for both deficiency groups compared with WT), along with plasma triglycerides (complete deficiency, 26 mg/dL; partial deficiency, 79 mg/dL; WT, 88 mg/dL; P<0.05 for both deficiency groups compared with WT) without any significant difference in hepatic fat (complete deficiency, 9.8%; partial deficiency, 10.1%; WT, 9.9%; P>0.05 for both deficiency groups compared with WT) or severity of hepatic steatosis as assessed by magnetic resonance imaging. In addition, ANGPTL3 deficiency did not alter the distribution of extrahepatic fat. Results from Mendelian randomization analyses in 36 703 participants from the UK Biobank demonstrated that genetically determined ANGPTL3 plasma protein levels were causally associated with low-density lipoprotein cholesterol (P=1.7×10-17) and triglycerides (P=3.2×10-18) but not with hepatic fat (P=0.22). CONCLUSIONS: ANGPTL3 deficiency related to LoF mutations in ANGPTL3, as well as genetically determined reduction of plasma ANGPTL3 levels, is not associated with hepatic steatosis. Therapeutic approaches to inhibit ANGPTL3 production in hepatocytes are not necessarily expected to result in the increased risk for hepatic steatosis that was observed with vupanorsen.

Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations.

Humoral immunity is sensitive to evasion by SARS-CoV-2 mutants, but CD8 T cells seem to be more resistant to mutational inactivation. By a systematic analysis of 30 spike variant peptides containing the most relevant VOC and VOI mutations that have accumulated overtime, we show that in vaccinated and convalescent subjects, mutated epitopes can have not only a neutral or inhibitory effect on CD8 T cell recognition but can also enhance or generate de novo CD8 T cell responses. The emergence of these mutated T cell function enhancing epitopes likely reflects an epiphenomenon of SARS-CoV-2 evolution driven by antibody evasion and increased virus transmissibility. In a subset of individuals with weak and narrowly focused CD8 T cell responses selection of these heteroclitic-like epitopes may bear clinical relevance by improving antiviral protection. The functional enhancing effect of these peptides is also worth of consideration for the future development of new generation, more potent COVID-19 vaccines.

Efficacy and safety of lomitapide in familial chylomicronaemia syndrome.

BACKGROUND AND AIMS: Familial chylomicronaemia syndrome (FCS) is a rare autosomal recessive disorder, resulting in elevated triglycerides (TGs), abdominal pain and pancreatitis. Treatment options are limited. Lomitapide, a microsomal triglyceride transfer protein inhibitor, is approved for the treatment of homozygous familial hypercholesterolaemia. Whether its therapeutic use may be extended to FCS remains unknown. The aim of this study was to evaluate the efficacy and safety of lomitapide in adult patients with FCS. METHODS: The open-label, single-arm 'LOCHNES' study of lomitapide in FCS enrolled patients >18 years with genetically confirmed FCS, elevated fasting TG ≥ 750 mg/dL and history of pancreatitis. Patients were administered lomitapide to the maximum tolerated dose for 26 weeks. The primary endpoint was the percent change in TGs from baseline to Week 26. RESULTS: Eighteen patients were enrolled with median baseline TG levels 1803.5 mg/dL (97.5% CI, 1452-2391 mg/dL). At Week 26, median fasting TGs were reduced to 305 mg/dL (97.5% CI 219-801 mg/dL; 70.5% reduction); median lomitapide dose was 35 mg/day; 13 patients achieved TGs ≤750 mg/dL. Adverse events were mild to moderate and mainly related to gastrointestinal tolerability. Liver imaging at baseline and Week 26 revealed hepatic fat increases from median 12.0%-32.5%, while median hepatic stiffness remained normal. No patient experienced acute pancreatitis or severe abdominal pain during lomitapide treatment. CONCLUSIONS: Lomitapide is effective and well tolerated in reducing TGs in FCS patients with a history of pancreatitis. Larger studies are warranted to determine lomitapide effectiveness in FCS.

Refinement of pathogenicity classification of variants associated with familial hypercholesterolemia: Implications for clinical diagnosis.

BACKGROUND: The lack of functional evidence for most variants detected during the molecular screening of patients with clinical familial hypercholesterolemia (FH) makes the definitive diagnosis difficult. METHODS: A total of 552 variants in LDLR, APOB, PCSK9 and LDLRAP1 genes found in 449 mutation-positive FH (FH/M+) patients were considered. Pathogenicity update was performed following the American College of Medical Genetics and Genomics (ACMG) guidelines with additional specifications on copy number variants, functional studies, in silico prediction and co-segregation criteria for LDLR, APOB and PCSK9 genes. Pathogenicity of LDLRAP1 variants was updated by using ACMG criteria with no change to original scoring. RESULTS: After reclassification, the proportion of FH/M+ carriers of pathogenic (P) or likely pathogenic (LP) variants, and FH/M+ carriers of likely benign (LB) or benign (B) variants, was higher than that defined by standard criteria (81.5% vs. 79.7% and 7.1% vs. 2.7%). The refinement of pathogenicity classification also reduced the percentage of FH with variants of uncertain significance (VUS) (17.7% vs. 11.4%). After adjustment, the FH diagnosis by refined criteria best predicted LDL-C levels (Padj <0.001). Notably, FH with VUS variants had higher LDL-C than those with LB (all Padj ≤ 0.033), but similar to those with LP variants. CONCLUSION: Accurate variant interpretation best predicts the increase of LDL-C levels and shows its clinical utility in the molecular diagnosis of FH.

Clinical Implications of Monogenic Versus Polygenic Hypercholesterolemia: Long-Term Response to Treatment, Coronary Atherosclerosis Burden, and Cardiovascular Events.

Background Familial hypercholesterolemia (FH) may arise from deleterious monogenic variants in FH-causing genes as well as from a polygenic cause. We evaluated the relationships between monogenic FH and polygenic hypercholesterolemia in influencing the long-term response to therapy and the risk of atherosclerosis. Methods and Results A cohort of 370 patients with clinically diagnosed FH were screened for monogenic mutations and a low-density lipoprotein-rising genetic risk score >0.69 to identify polygenic cause. Medical records were reviewed to estimate the response to lipid-lowering therapies and the occurrence of major atherosclerotic cardiovascular events during a median follow-up of 31.0 months. A subgroup of patients (n=119) also underwent coronary computed tomographic angiography for the evaluation of coronary artery calcium score and severity of coronary stenosis as compared with 135 controls. Two hundred nine (56.5%) patients with hypercholesterolemia were classified as monogenic (FH/M+), 89 (24.1%) as polygenic, and 72 (19.5%) genetically undefined (FH/M-). The response to lipid-lowering therapy was poorest in monogenic, whereas it was comparable in patients with polygenic hypercholesterolemia and genetically undetermined. Mean coronary artery calcium score and the prevalence of coronary artery calcium >100 units were significantly higher in FH/M+ as compared with both FH/M- and controls. Finally, after adjustments for confounders, we observed a 5-fold higher risk of incident major atherosclerotic cardiovascular events in FH/M+ (hazard ratio, 4.8; 95% CI, 1.06-21.36; Padj=0.041). Conclusions Monogenic cause of FH is associated with lower response to conventional cholesterol-lowering therapies as well as with increased burden of coronary atherosclerosis and risk of atherosclerotic-related events. Genetic testing for hypercholesterolemia is helpful in providing important prognostic information.

Evolving trend in the management of heterozygous familial hypercholesterolemia in Italy: A retrospective, single center, observational study.

BACKGROUND AND AIMS: The effective reduction of LDL-C in patients with heterozygous familial hypercholesterolemia (HeFH) is crucial to reduce their increased cardiovascular risk. Diagnostic and therapeutic (PCSK9 inhibitors) tools to manage HeFH improved in recent years. However, the impact of these progresses in ameliorating the contemporary real-world care of these patients remains to be determined. Aim of this study was to assess the evolution of treatments and LDL-C control in a cohort of HeFH patients in Italy. METHODS AND RESULTS: Four hundred six clinically diagnosed HeFH followed in a single, tertiary lipid centre were included in this survey. Data on lipid levels and medications were collected at baseline and during a median 3-year follow-up. At baseline, 19.8% of patients were receiving conventional high-potency lipid lowering therapies (LLT) and this percentage increased up to 50.8% at last visit. The knowledge of results of molecular diagnosis was associated with a significant increase in treatment intensity and LDL-C lowering. Nevertheless, the new LDL-C target (<70 mg/dl) was achieved only in 3.6% of HeFH patients under conventional LLTs and this proportion remained low (2.9%) also in those exposed to maximal conventional LLT. In 51 patients prescribed with PCSK9 inhibitors, 64.6% and 62.1% reached LDL-C<70 mg/dl at 3- and 12-month follow-up, respectively. CONCLUSIONS: Although treatments of HeFH improved over time, LDL-C target achievement with conventional LLT remains poor, mainly among women. The use of molecular diagnosis and even more the prescription of PCSK9i may improve LDL-C control in these patients.

Spectrum of Mutations and Long-Term Clinical Outcomes in Genetic Chylomicronemia Syndromes.

OBJECTIVE: Familial chylomicronemia syndrome (FCS) and multifactorial chylomicronemia syndrome (MCS) are the prototypes of monogenic and polygenic conditions underlying genetically based severe hypertriglyceridemia. These conditions have been only partially investigated so that a systematic comparison of their characteristics remains incomplete. We aim to compare genetic profiles and clinical outcomes in FCS and MCS. Approach and Results: Thirty-two patients with severe hypertriglyceridemia (triglyceride >1000 mg/dL despite lipid-lowering treatments with or without history of acute pancreatitis) were enrolled. Rare and common variants were screened using a panel of 18 triglyceride-raising genes, including the canonical LPL, APOC2, APOA5, GP1HBP1, and LMF1. Clinical information was collected retrospectively for a median period of 44 months. Across the study population, 37.5% were classified as FCS due to the presence of biallelic, rare mutations and 59.4% as MCS due to homozygosity for nonpathogenic or heterozygosity for pathogenic variants in canonical genes, as well as for rare and low frequency variants in noncanonical genes. As compared with MCS, FCS patients showed a lower age of hypertriglyceridemia onset, higher levels of on-treatment triglycerides, and 3-fold higher incidence rate of acute pancreatitis. CONCLUSIONS: Our data indicate that the genetic architecture and natural history of FCS and MCS are different. FCS expressed the most severe clinical phenotype as determined by resistance to triglyceride-lowering medications and higher incidence of acute pancreatitis episodes. The most common genetic abnormality underlying FCS was represented by biallelic mutations in LPL while APOA5 variants, in combination with high rare polygenic burden, were the most frequent genotype of MCS.

A novel splicing mutation in the ABCA1 gene, causing Tangier disease and familial HDL deficiency in a large family.

Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene. ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G > A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86 ±â€¯0.16 mmol/L and 92.2 ±â€¯10.9 mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G > A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency. Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.

Evaluation of Polygenic Determinants of Non-Alcoholic Fatty Liver Disease (NAFLD) By a Candidate Genes Resequencing Strategy.

NAFLD is a polygenic condition but the individual and cumulative contribution of identified genes remains to be established. To get additional insight into the genetic architecture of NAFLD, GWAS-identified GCKR, PPP1R3B, NCAN, LYPLAL1 and TM6SF2 genes were resequenced by next generation sequencing in a cohort of 218 NAFLD subjects and 227 controls, where PNPLA3 rs738409 and MBOAT7 rs641738 genotypes were also obtained. A total of 168 sequence variants were detected and 47 were annotated as functional. When all functional variants within each gene were considered, only those in TM6SF2 accumulate in NAFLD subjects compared to controls (P = 0.04). Among individual variants, rs1260326 in GCKR and rs641738 in MBOAT7 (recessive), rs58542926 in TM6SF2 and rs738409 in PNPLA3 (dominant) emerged as associated to NAFLD, with PNPLA3 rs738409 being the strongest predictor (OR 3.12, 95% CI, 1.8-5.5, P < 0.001). A 4-SNPs weighted genetic risk score value >0.28 was associated with a 3-fold increased risk of NAFLD. Interestingly, rs61756425 in PPP1R3B and rs641738 in MBOAT7 genes were predictors of NAFLD severity. Overall, TM6SF2, GCKR, PNPLA3 and MBOAT7 were confirmed to be associated with NAFLD and a score based on these genes was highly predictive of this condition. In addition, PPP1R3B and MBOAT7 might influence NAFLD severity.

Circulating miR-200c is up-regulated in paediatric patients with familial hypercholesterolaemia and correlates with miR-33a/b levels: implication of a ZEB1-dependent mechanism.

Hypercholesterolaemia provokes reactive oxygen species (ROS) increase and is a major risk factor for cardiovascular disease (CVD) development. We previously showed that circulating miR-33a/b expression levels were up-regulated in children with familial hypercholesterolaemia (FH). miR-33a/b control cholesterol homoeostasis and recently miR-33b has been demonstrated to directly target the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1). The latter acts in a negative feedback loop with the miR-200 family. Our previous studies showed that the ROS-dependent miR-200c up-regulation induces endothelial dysfunction and provokes a ZEB1-dependent apoptosis and senescence. In the present study, we aimed to verify whether circulating miR-200c was induced in FH children, and whether a correlation existed with miR-33a/b Total RNA was extracted from plasma of 28 FH children and 25 age-matched healthy subjects (HS) and miR-200c levels were measured. We found that miR-200c was up-regulated in FH compared with HS (4.00 ± 0.48-fold increase, P<0.05) and exhibited a positive correlation with miR-33a/b. miR-200c did not correlate with plasma lipids, but correlated with C-reactive protein (CRP) plasma levels and glycaemia (GLI). Ordinary least squares (OLS) regression analysis revealed that miR-200c was significantly affected by GLI and by miR-33a (P<0.01; P<0.001 respectively). Moreover, we found that miR-33 overexpression, in different cell lines, decreased ZEB1 expression and up-regulated both the intracellular and the extracellular miR-200c expression levels. In conclusion, circulating miR-200c is up-regulated in FH, probably due to oxidative stress and inflammation and via a miR-33a/b-ZEB1-dependent mechanism. The present study could provide the first evidence to point to the use of miR-33a/b and miR-200c, as early biomarkers of CVD, in paediatric FH.

Analysis of Children and Adolescents with Familial Hypercholesterolemia.

OBJECTIVE: To evaluate the effectiveness of criteria based on child-parent assessment in predicting familial hypercholesterolemia (FH)-causative mutations in unselected children with hypercholesterolemia. STUDY DESIGN: LDLR, APOB, and PCSK9 genes were sequenced in 78 children and adolescents (mean age 8.4 ± 3.7 years) with clinically diagnosed FH. The presence of polygenic hypercholesterolemia was further evaluated by genotyping 6 low-density lipoprotein cholesterol (LDL-C)-raising single-nucleotide polymorphisms. RESULTS: Thirty-nine children (50.0%) were found to carry LDLR mutant alleles but none with APOB or PCSK9 mutant alleles. Overall, 27 different LDLR mutations were identified, and 2 were novel. Children carrying mutations showed higher LDL-C (215.2 ± 52.7 mg/dL vs 181.0 ± 44.6 mg/dL, P <.001) and apolipoprotein B levels (131.6 ± 38.3 mg/dL vs 100.3 ± 30.0 mg/dL, P <.004), compared with noncarriers. A LDL-C of ~190 mg/dL was the optimal value to discriminate children with and without LDLR mutations. When different diagnostic criteria were compared, those proposed by the European Atherosclerosis Society showed a reasonable balance between sensitivity and specificity in the identification of LDLR mutations. In children without mutation, the FH phenotype was not caused by the aggregation of LDL-C raising single-nucleotide polymorphisms. CONCLUSIONS: In unselected children with hypercholesterolemia, LDL-C levels >190 mg/dL and a positive family history of hypercholesterolemia appeared to be the most reliable criteria for detecting FH. As 50% of children with suspected FH did not carry FH-causing mutations, genetic testing should be considered.

Non-alcoholic fatty liver disease and subclinical atherosclerosis: A comparison of metabolically- versus genetically-driven excess fat hepatic storage.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is frequently associated with atherosclerosis. However, it is unclear whether this association is related to excess fat liver storage per se or to metabolic abnormalities that typically accompany NAFLD. To investigate this, we compared individuals with hepatic steatosis driven by metabolic disturbances to those with hepatic steatosis associated with the rs738409 GG genotype in the patatin-like phospholipase domain-containing 3 gene (PNPLA3). METHODS: Carotid intima-media thickness (CIMT), as a surrogate marker of subclinical atherosclerosis, was measured in 83 blood donors with the mutant GG genotype (group G), 100 patients with features of metabolic syndrome (MetS) but the wildtype CC genotype (group M), and 74 blood donors with the wildtype CC genotype (controls). Fatty liver was evaluated by ultrasonography and hepatic fat fraction (HFF) was measured using magnetic resonance (MRS/MRI) in 157 subjects. RESULTS: Compared with group G and controls, group M subjects were older and had increased adiposity indices, dyslipidemia, insulin resistance and elevated transaminase levels (all p < 0.05). They also had a more fatty liver on both ultrasonography and MRS/MRI. After adjustment for confounders (including severity of hepatic steatosis), the median CIMT in group M (0.84 [0.70-0.95] mm) was significantly greater than that in group G (0.66 [0.55-0.74] mm; p < 0.001), which was similar to that in controls (0.70 [0.64-0.81] mm). Results were similar in the subgroup evaluated using MRS/MRI. CONCLUSIONS: Excess liver fat accumulation appeared to increase the burden of subclinical atherosclerosis only when it is associated with metabolic abnormalities.

Effects of angiopoietin-like protein 3 deficiency on postprandial lipid and lipoprotein metabolism.

The consequences of angiopoietin-like protein 3 (ANGPTL3) deficiency on postprandial lipid and lipoprotein metabolism has not been investigated in humans. We studied 7 homozygous (undetectable circulating ANGPTL3 levels) and 31 heterozygous (50% of circulating ANGPTL3 levels) subjects with familial combined hypolipidemia (FHBL2) due to inactivating ANGPTL3 mutations in comparison with 35 controls. All subjects were evaluated at fasting and during 6 h after a high fat meal. Postprandial lipid and lipoprotein changes were quantified by calculating the areas under the curve (AUCs) using the 6 h concentration data. Plasma changes of ß-hydroxybutyric acid (ß-HBA) were measured as marker of hepatic oxidation of fatty acids. Compared with controls, homozygotes showed lower incremental AUCs (iAUCs) of total TG (-69%, P < 0.001), TG-rich lipoproteins (-90%, P < 0.001), apoB-48 (-78%, P = 0.032), and larger absolute increase of FFA (128%, P < 00.1). Also, heterozygotes displayed attenuated postprandial lipemia, but the difference was significant only for the iAUC of apoB-48 (-28%; P < 0.05). During the postprandial period, homozygotes, but not heterozygotes, showed a lower increase of ß-HBA. Our findings demonstrate that complete ANGPTL3 deficiency associates with highly reduced postprandial lipemia probably due to faster catabolism of intestinally derived lipoproteins, larger expansion of the postprandial FFA pool, and decreased influx of dietary-derived fatty acids into the liver. These results add information on mechanisms underlying hypolipidemia in FHBL2.

Contribution of mutations in low density lipoprotein receptor (LDLR) and lipoprotein lipase (LPL) genes to familial combined hyperlipidemia (FCHL): a reappraisal by using a resequencing approach.

BACKGROUND: Defective low-density lipoprotein receptor (LDLR) and lipoprotein lipase (LPL) alleles have been implicated in familial combined hyperlipidemia (FCHL). However, their contribution might have been influenced by diagnostic criteria. This study was aimed to reassess the frequency of rare and common variants in LDLR and LPL in FCHL individuals classified with stringent criteria. METHODS: LDLR and LPL were resequenced in 208 FHCL and 171 controls. Variants were classified as loss- (LOF) or gain-of-function (GOF) based upon in silico prediction, familial segregation and available functional data. RESULTS: Eight LOF variants were detected in LDLR, 6 of which were missense and 2 were predicted to disrupt normal splicing; all were present at heterozygous state. They were found in 10 FCHL but not in controls, thus indicating that 4.8% of FCHL individuals should be reclassified as FH. LDL-C (positive) and BMI (negative) were the strongest predictors of LDLR mutations with LDL-C 181 mg/dl being the best threshold for diagnosing the presence of dysfunctional LDLR alleles. The cumulative prevalence of definite LPL defective alleles (1 rare and 2 common heterozygous missense variants) was comparable between FCHL and controls (10.1% vs. 10.5%). Conversely, the LPL GOF variant p.Ser474* showed a lower frequency in FCHL than in controls (13.5% vs. 24.0%, p = 0.008). Overall, LOF LPL variants did not show a TG-modulating effect. CONCLUSIONS: Our findings indicate that, in well characterized FCHL individuals, variants in LDLR and LPL provide a small contribution to this dyslipidemia, thus limiting the need for such genetic testing.

Circulating miR-33a and miR-33b are up-regulated in familial hypercholesterolaemia in paediatric age.

Hypercholesterolaemia is one of the major causes of CVD (cardiovascular disease). It is associated with enhanced oxidative stress, leading to increased lipid peroxidation which in turn determines endothelial dysfunction and susceptibility to coronary vasoconstriction and atherosclerosis. Different miRNAs are involved in the pathogenesis of CVD and play an important role in inflammatory process control, therefore, together with atherogenic factors, they can stimulate atherosclerotic degeneration of the vessel walls of arteries. miR-33a and miR-33b play a pivotal role in a variety of biological processes including cholesterol homoeostasis, HDL (high-density lipoprotein)-cholesterol formation, fatty acid oxidation and insulin signalling. Our study aimed to determine whether circulating miR-33a and miR-33b expression was altered in familial hypercholesterolaemic children. Total RNA was extracted from plasma, and miR-33a and miR-33b were measured by quantitative real-time PCR. We found that miR-33a and miR-33b were significantly up-regulated in the plasma of 28 hypercholesterolaemic children compared with 25 healthy subjects (4.49±0.27-fold increase, P<0.001, and 3.21±0.39-fold increase, P<0.05 respectively), and for both miRNAs, a positive correlation with total cholesterol, LDL (low-density lipoprotein)-cholesterol, LDL-cholesterol/HDL-cholesterol ratio, apolipoprotein B, CRP (C-reactive protein) and glycaemia was found. OLS (ordinary least squares) regression analysis revealed that miR-33a was significantly affected by the presence of FH (familial hypercholesterolaemia), glycaemia and CRP (P<0.001, P<0.05 and P<0.05 respectively). The same analysis showed that miR-33b was significantly related to FH and CRP (P<0.05 and P<0.05 respectively). Although it is only explorative, the present study could be the first to point to the use of miR-33a and miR-33b as early biomarkers for cholesterol levels in childhood, once validated in independent larger cohorts.

Correction: Atherogenic Dyslipidemia in Children: Evaluation of Clinical, Biochemical and Genetic Aspects.

[This corrects the article DOI: 10.1371/journal.pone.0120099.].

Atherogenic dyslipidemia in children: evaluation of clinical, biochemical and genetic aspects.

The precursors of atherogenic dyslipidemia (AD) are not well defined. Therefore, we investigated 62 non-obese, non-diabetic AD and 221 normolipemic children. Anthropometric parameters, blood pressure and biochemical measures were obtained in index children, their parents and all available siblings. The heritability (h(2)) of anthropometric and biochemical traits was estimated by SOLAR. Rare and common variants in APOA1 and LPL genes were screened by re-sequencing. Compared to normolipemic, AD children showed increased body mass index, waist circumference, plasma glucose, insulin, ApoB, HOMA-IR, hs-CRP and lower adiponectin (p<0.001 for all). Metabolic syndrome was present in 40% of AD while absent in controls. All traits (except adiponectin and hs-CRP) showed a strong familial aggregation, with plasma glucose having the highest heritability (89%). Overall, 4 LPL loss-of-function mutations were detected (p.Asp9Asn, p.Ser45Asn, p.Asn291Ser, p.Leu365Val) and their cumulative prevalence was higher in AD than in control children (0.073 vs. 0.026; P=0.038). The LPL p.S447* gain-of-function mutation, resulted to be less frequent in AD than in control children (0.064 vs. 0.126; P=0.082). No variant in the APOA1 gene was found. Our data indicate that AD is a rather common dyslipidemia in childhood; it associates with metabolic abnormalities typical of insulin resistant state and shows a strong familial aggregation. LPL variants may contribute to the development of AD phenotype.