Unraveling the genetic background of individuals with a clinical familial hypercholesterolemia phenotype.

Familial hypercholesterolemia (FH) is a common genetic disorder of lipid metabolism caused by pathogenic/likely pathogenic variants in LDLR, APOB, and PCSK9 genes. Variants in FH-phenocopy genes (LDLRAP1, APOE, LIPA, ABCG5, and ABCG8), polygenic hypercholesterolemia, and hyperlipoprotein (a) [Lp(a)] can also mimic a clinical FH phenotype. We aim to present a new diagnostic tool to unravel the genetic background of clinical FH phenotype. Biochemical and genetic study was performed in 1,005 individuals with clinical diagnosis of FH, referred to the Portuguese FH Study. A next-generation sequencing panel, covering eight genes and eight SNPs to determine LDL-C polygenic risk score and LPA genetic score, was validated, and used in this study. FH was genetically confirmed in 417 index cases: 408 heterozygotes and 9 homozygotes. Cascade screening increased the identification to 1,000 FH individuals, including 11 homozygotes. FH-negative individuals (phenotype positive and genotype negative) have Lp(a) >50 mg/dl (30%), high polygenic risk score (16%), other monogenic lipid metabolism disorders (1%), and heterozygous pathogenic variants in FH-phenocopy genes (2%). Heterozygous variants of uncertain significance were identified in primary genes (12%) and phenocopy genes (7%). Overall, 42% of our cohort was genetically confirmed with FH. In the remaining individuals, other causes for high LDL-C were identified in 68%. Hyper-Lp(a) or polygenic hypercholesterolemia may be the cause of the clinical FH phenotype in almost half of FH-negative individuals. A small part has pathogenic variants in ABCG5/ABCG8 in heterozygosity that can cause hypercholesterolemia and should be further investigated. This extended next-generation sequencing panel identifies individuals with FH and FH-phenocopies, allowing to personalize each person's treatment according to the affected pathway.

Generation and validation of a classification model to diagnose familial hypercholesterolaemia in adults.

BACKGROUND AND AIMS: The early diagnosis of familial hypercholesterolaemia is associated with a significant reduction in cardiovascular disease (CVD) risk. While the recent use of statistical and machine learning algorithms has shown promising results in comparison with traditional clinical criteria, when applied to screening of potential FH cases in large cohorts, most studies in this field are developed using a single cohort of patients, which may hamper the application of such algorithms to other populations. In the current study, a logistic regression (LR) based algorithm was developed combining observations from three different national FH cohorts, from Portugal, Brazil and Sweden. Independent samples from these cohorts were then used to test the model, as well as an external dataset from Italy. METHODS: The area under the receiver operating characteristics (AUROC) and precision-recall (AUPRC) curves was used to assess the discriminatory ability among the different samples. Comparisons between the LR model and Dutch Lipid Clinic Network (DLCN) clinical criteria were performed by means of McNemar tests, and by the calculation of several operating characteristics. RESULTS: AUROC and AUPRC values were generally higher for all testing sets when compared to the training set. Compared with DLCN criteria, a significantly higher number of correctly classified observations were identified for the Brazilian (p < 0.01), Swedish (p < 0.01), and Italian testing sets (p < 0.01). Higher accuracy (Acc), G mean and F1 score values were also observed for all testing sets. CONCLUSIONS: Compared to DLCN criteria, the LR model revealed improved ability to correctly classify observations, and was able to retain a similar number of FH cases, with less false positive retention. Generalization of the LR model was very good across all testing samples, suggesting it can be an effective screening tool if applied to different populations.

The Hypocholesterolemic Potential of the Edible Algae Fucus vesiculosus: Proteomic and Quantitative PCR Analysis.

A brown seaweed consumed worldwide, Fucus vesiculosus, has been used to prevent atherosclerosis and hypercholesterolemia, among other uses. However, the mechanisms of action that lead to these effects are not yet fully understood. This work aims to study the in vitro effect of an aqueous extract of F. vesiculosus, previously characterized as rich in phlorotannins and peptides, on the expression of different proteins involved in the synthesis and transport of cholesterol. A proteomic analysis, Western blot, and qRT-PCR analysis were performed to identify protein changes in HepG2 cells exposed to 0.25 mg/mL of the F. vesiculosus extract for 24 h. The proteomic results demonstrated that, in liver cells, the extract decreases the expression of four proteins involved in the cholesterol biosynthesis process (CYP51A1, DHCR24, HMGCS1 and HSD17B7). Additionally, a 12.76% and 18.40% decrease in the expression of two important transporters proteins of cholesterol, NPC1L1 and ABCG5, respectively, was also observed, as well as a 30% decrease in NPC1L1 mRNA levels in the cells exposed to the extract compared to control cells. Our study reveals some of the mechanisms underlying the actions of bioactive compounds from F. vesiculosus that may explain its previously reported hypocholesterolemic effect, future prospecting its use as a functional food.

Comparative study on the performance of different classification algorithms, combined with pre- and post-processing techniques to handle imbalanced data, in the diagnosis of adult patients with familial hypercholesterolemia.

Familial Hypercholesterolemia (FH) is an inherited disorder of cholesterol metabolism. Current criteria for FH diagnosis, like Simon Broome (SB) criteria, lead to high false positive rates. The aim of this work was to explore alternative classification procedures for FH diagnosis, based on different biological and biochemical indicators. For this purpose, logistic regression (LR), naive Bayes classifier (NB), random forest (RF) and extreme gradient boosting (XGB) algorithms were combined with Synthetic Minority Oversampling Technique (SMOTE), or threshold adjustment by maximizing Youden index (YI), and compared. Data was tested through a 10 × 10 repeated k-fold cross validation design. The LR model presented an overall better performance, as assessed by the areas under the receiver operating characteristics (AUROC) and precision-recall (AUPRC) curves, and several operating characteristics (OC), regardless of the strategy to cope with class imbalance. When adopting either data processing technique, significantly higher accuracy (Acc), G-mean and F1 score values were found for all classification algorithms, compared to SB criteria (p < 0.01), revealing a more balanced predictive ability for both classes, and higher effectiveness in classifying FH patients. Adjustment of the cut-off values through pre or post-processing methods revealed a considerable gain in sensitivity (Sens) values (p < 0.01). Although the performance of pre and post-processing strategies was similar, SMOTE does not cause model's parameters to loose interpretability. These results suggest a LR model combined with SMOTE can be an optimal approach to be used as a widespread screening tool.

Functional profiling of LDLR variants: Important evidence for variant classification: Functional profiling of LDLR variants.

BACKGROUND: Familial Hypercholesterolemia (FH) is a semidominant disorder of the lipid metabolism associated with premature atherosclerosis and coronary heart disease. So far, about 3,000 unique LDLR variants have been described, most of which lack functional evidence proving their effect on LDLR function, despite the important role that functional studies play in variant classification. OBJECTIVE: In this work, we aimed to functionally characterize 13 rare missense variants, identified worldwide and in Portugal, in clinical FH patients. METHODS: LDLR-deficient CHO-ldlA7 cells were transfected with plasmids carrying different LDLR variants generated by site-directed mutagenesis. LDLR activity and expression were assessed by FACS. RESULTS: 11/13 variants affect LDLR function (p.Cys109Phe; p.Cys143Arg; p.Glu267Lys; p.Cys352Ser; p.Ile451Thr; p.His485Gln; p.Asp492Asn; p.Val500Ala; p.Gly529Arg; p.Phe614Ile; p.Glu626Lys) and 2/13 are inconclusive (p.Arg81Cys; p.Gly98Arg;). CONCLUSION: Of the 13 variants studied, 8 were classified as VUS by ACMG criteria, but for 7 of these 8, our functional studies were able to reassign them as Likely pathogenic or Pathogenic. For an accurate diagnosis, an effort must be made to improve functional characterization of putative disease-causing variants.

Performance comparison of different classification algorithms applied to the diagnosis of familial hypercholesterolemia in paediatric subjects.

Familial Hypercholesterolemia (FH) is an inherited disorder of lipid metabolism, characterized by increased low density lipoprotein cholesterol (LDLc) levels. The main purpose of the current work was to explore alternative classification methods to traditional clinical criteria for FH diagnosis, based on several biochemical and biological indicators. Logistic regression (LR), decision tree (DT), random forest (RF) and naive Bayes (NB) algorithms were developed for this purpose, and thresholds were optimized by maximization of Youden index (YI). All models presented similar accuracy (Acc), specificity (Spec) and positive predictive values (PPV). Sensitivity (Sens) and G-mean values were significantly higher in LR and RF models, compared to the DT. When compared to Simon Broome (SB) biochemical criteria for FH diagnosis, all models presented significantly higher Acc, Spec and G-mean values (p < 0.01), and lower negative predictive value (NPV, p < 0.05). Moreover, LR and RF models presented comparable Sens values. Adjustment of the cut-off point by maximizing YI significantly increased Sens values, with no significant loss in Acc. The obtained results suggest such classification algorithms can be a viable alternative to be used as a widespread screening method. An online application has been developed to assess the performance of the LR model in a wider population.

Rare primary dyslipidaemias associated with low LDL and HDL cholesterol values in Portugal.

Background: Dyslipidaemia represents a group of disorders of lipid metabolism, characterized by either an increase or decrease in lipid particles, usually associated with triglycerides, LDL cholesterol (LDL-C) and/or HDL cholesterol (HDL-C). Most hyperlipidaemias and HDL deficiencies confer an increased cardiovascular risk, while hypolipidaemia, such as abeta or hypobetalipoproteinemia, may present different manifestations ranging from poor weight progression to neurological manifestations. The aim of this study is to present 7 cases with rare dyslipidaemias associated with low LDL or low HDL cholesterol values, referred to our laboratory for the genetic identification of the cause of the dyslipidaemia. Methods: Lipid profile was determined for each individual in an automated equipment Integra Cobas (Roche). Molecular analysis was performed by NGS with a target panel of 57 genes involved in lipid metabolism (Sure select QXT, Agilent) and samples were run in a NextSEQ Sequencer (Illumina). Only genes associated to rare forms of low HDL-c or LDL-c were analysed for this work, namely: ABCA1, APOA1, LCAT, SCARB1, APOB, PCSK9, MTTP, SAR1B, and ANGPTL3. All rare variants (MAF<5%) found in these genes were confirmed by Sanger sequencing. Results and discussion: This study includes 7 index cases (IC), with the following clinical diagnoses: Fish Eye Disease (1), Hypoalphalipoproteinemia (1) and Abetalipoproteinemia (ABL) / Familial Hypobetalipoproteinemia (FHBL) (5). We have identified one IC with a compound heterozygosity in LCAT causing Fish Eye Disease and one IC with a variant in ABCA1 in homozygosity causing Tangier disease. We found variants causing homozygous FHBL in 2 IC, one of whom has an undescribed pathogenic variant in homozygosity in APOB (c.12087+1G>A) and the other is a possible compound heterozygous for APOB variants c.2604+1G>A and c.4651C>T/p.(Gln1551*). In two patients only a variant in heterozygosity (c.3365delG/p.(Gly1122Vfs*62) and c.11095A>T/p.(Arg3699*)). In the remaining patient, no variants were identified. NGS proved to be a fundamental key for genetic testing of rare lipid disorders, allowing us to find the genetic cause of disease in 6/7 patients with low HDL-c and LDL-c. Patients with these rare conditions should be identified as early as possible in order to minimize or prevent clinical manifestations. The unsolved case is still under investigation.

Applicability of Martin-Hopkins formula and comparison with Friedewald formula for estimated low-density lipoprotein cholesterol in e_COR study population.

INTRODUCTION: Low-density lipoprotein cholesterol (LDL) is essential in managing cardiovascular disease risk. Since 1972, the Friedewald formula has been used to estimate LDL concentration, although with some limitations. In 2013, Martin et al. proposed a similar but more accurate formula for calculating LDL. AIM: To assess the applicability of the new formula, which we have named the Martin-Hopkins formula, in the Portuguese population and compare it with the Friedewald formula using direct LDL. METHODS: Cross-sectional study, including 1689 participants from the e_COR study. We applied the Martin-Hopkins and Friedewald formulas for estimated LDL (LDL-M and LDL-F). The Friedewald formula was not applied in 12 cases due to triglycerides ≥400mg/dL. Direct LDL was measured and the accepted significance level was p<0.05. RESULTS: Of the total subjects, 50.2% were male and had a median age of 51 (34) years. LDL-D was 117.0 (44.0) mg/dL, LDL-M was 114.6 (43.7) mg/dL and LDL-F was 113.8 (43.2) mg/dL. The Spearman coefficient (ρ) between LDL-M/LDL-D was 0.987 and between LDL-F/LDL-D was 0.983, p=0.001. This strong correlation was maintained in the group with diabetes (LDL-M/LDL-D ρ=0.987; LDL-F/LDL-D ρ=0.978, p=0.001) and hypertriglyceridemia (LDL-M/LDL-D ρ=0.983; LDL-F/LDL-D ρ=0.982, p=0.001). In terms of agreement, the highest value of κ=0.90 was obtained for LDL-M when LDL-D <100mg/dL. CONCLUSION: The Martin-Hopkins formula performed well and had good applicability, showing superiority in relation to the Friedewald formula, especially for LDL-D values <100mg/dL, diabetes, and hypertriglyceridemia.

Characterization of Two Variants at Met 1 of the Human LDLR Gene Encoding the Same Amino Acid but Causing Different Functional Phenotypes.

Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, characterized by increased levels of total and LDL plasma cholesterol, which leads to premature atherosclerosis and coronary heart disease. FH phenotype has considerable genetic heterogeneity and phenotypic variability, depending on LDL receptor activity and lifestyle. To improve diagnosis and patient management, here, we characterized two single nucleotide missense substitutions at Methionine 1 of the human LDLR gene (c.1A>T/p.(Met1Leu) and c.1A>C/p.(Met1Leu)). We used a combination of Western blot, flow cytometry, and luciferase assays to determine the effects of both variants on the expression, activity, and synthesis of LDLR. Our data show that both variants can mediate translation initiation, although the expression of variant c.1A>T is very low. Both variants are in the translation initiation codon and codify for the same amino acid p.(Met1Leu), yet they lead to different levels of impairment on LDLR expression and activity, corroborating different efficiencies of the translation initiation at these non-canonical initiation codons. The functional data of these variants allowed for an improved American College of Medical Genetics (ACMG) classification for both variants, which can allow a more personalized choice of the lipid-lowering treatment and dyslipidemia management, ultimately improving patients' prognosis.

Applicability of Martin-Hopkins formula and comparison with Friedewald formula for estimated low-density lipoprotein cholesterol in e_COR study population. / Aplicabilidade da fórmula Martin­Hopkins e comparação com a fórmula Friedewald na estimativa do colesterol LDL na população do estudo e_COR.

INTRODUCTION: Low-density lipoprotein cholesterol (LDL) is essential in managing cardiovascular disease risk. Since 1972, the Friedewald formula has been used to estimate LDL concentration, although with some limitations. In 2013, Martin et al. proposed a similar but more accurate formula for calculating LDL. AIM: To assess the applicability of the new formula, which we have named the Martin-Hopkins formula, in the Portuguese population and compare it with the Friedewald formula using direct LDL. MATERIAL AND METHODS: Cross-sectional study, including 1689 participants from the e_COR study. We applied the Martin-Hopkins and Friedewald formulas for estimated LDL (LDL-M and LDL-F). The Friedewald formula was not applied in 12 cases due to triglycerides ≥400mg/dL. Direct LDL was measured and the accepted significance level was p<0.05. RESULTS: Of the total subjects, 50.2% were male and had a median age of 51 (34) years. LDL-D was 117.0 (44.0) mg/dL, LDL-M was 114.6 (43.7) mg/dL and LDL-F was 113.8 (43.2) mg/dL. The Spearman coefficient (ρ) between LDL-M/LDL-D was 0.987 and between LDL-F/LDL-D was 0.983, p=0.001. This strong correlation was maintained in the group with diabetes (LDL-M/LDL-D ρ=0.987; LDL-F/LDL-D ρ=0.978, p=0.001) and hypertriglyceridemia (LDL-M/LDL-D ρ=0.983; LDL-F/LDL-D ρ=0.982, p=0.001). In terms of agreement, the highest value of κ=0.90 was obtained for LDL-M when LDL-D <100 mg/dL. CONCLUSION: The Martin-Hopkins formula performed well and had good applicability, showing superiority in relation to the Friedewald formula, especially for LDL-D values <100 mg/dL, diabetes, and hypertriglyceridemia.

LDLR variants functional characterization: Contribution to variant classification.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is an autosomal disorder of lipid metabolism presenting with increased cardiovascular risk. LDLR mutations are the cause of disease in 90% of the cases but functional studies have only been performed for about 15% of all LDLR variants. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 142 unique LDLR alterations were identified and 44 (30%) lack functional characterization. The aim of the present work is to increase evidence for variant classification by performing functional characterization of 13 LDLR missense alterations found in Portugal and in 20 other countries. METHODS: Different LDLR mutants were generated by site-directed mutagenesis and expressed in CHO-ldlA7 cells lacking endogenous expression of LDLR. To determine the effects of alterations on LDLR function, cell surface expression, binding and uptake of FITC-LDL were assessed by flow cytometry and Western blot. RESULTS: Of the 13 variants studied 7 were shown to affect LDLR function - expression, binding or uptake, with rates lower than 60%: p.(Cys184Tyr), p.(Gly207_Ser213del); p.(His211Asp); p.(Asp221Tyr); p.(Glu288Lys); p.(Gly592Glu) and p.(Asp601Val)). The remaining 6 variants do not alter the LDLR function. CONCLUSIONS: These studies contributed to an update of these variants classification: from the 9 variants classified as variants of unknown significance, 7 have reached now a final classification and 3 variants have improved classification from likely pathogenic to pathogenic. In Portugal, an additional 55 patients received an FH definite diagnosis thanks to these studies. Since only likely pathogenic and pathogenic variants are clinically actionable, this work shows the importance of functional studies for variant classification.

Metabolic Dysfunction and Asthma: Current Perspectives.

The increasing knowledge of the mechanisms involved in metabolism is shifting the paradigms by which the pathophysiology of many pulmonary diseases is understood. Metabolic dysfunction is recognized in obesity-associated asthma, but other metabolic conditions have been shown to be independently related to asthma. Novel insights have also recently been brought by metabolomics in this filed. The purpose of this review is to discuss current perspectives regarding metabolic dysfunction in asthma, from obesity-related asthma to other metabolic conditions and the role of current pharmacological therapeutic strategies and lifestyle interventions. Obesity is a well-recognized risk factor for asthma across the lifespan, which is generally associated with poorer response to current available treatments, rendering a more severe, refractory disease status. Besides the epidemiological and clinical link, untargeted metabolomics studies have recently supported the obesity-associated asthma phenotype at the molecular level. Not only obesity-related, but also other aspects of metabolic dysregulation can be independently linked to asthma. These include hyperinsulinemia, dyslipidemia and hypertension, which need to be taken into account, even in the non-obese patient. Untargeted metabolomics studies have further highlighted several other metabolic pathways that can be altered in asthma, namely regarding oxidative stress and systemic inflammation, and also suggesting the importance of microbiota in asthma pathogenesis. Considering the reduced response to corticosteroids, other pharmacologic treatments have been shown to be effective regardless of body mass index. Non-pharmacologic treatments (namely weight reduction and dietary changes) may bring substantial benefit to the asthmatic patient. Taken together, this evidence points towards the need to improve our knowledge in this filed and, in particular, to address the influence of environmental factors in metabolic dysfunction and asthma development. Personalized medicine is definitely needed to optimize treatment, including a holistic view of the asthmatic patient in order to set accurate pharmacologic therapy together with dietary, physical exercise and lifestyle interventions.

Phenotypical, Clinical, and Molecular Aspects of Adults and Children With Homozygous Familial Hypercholesterolemia in Iberoamerica.

OBJECTIVE: Characterize homozygous familial hypercholesterolemia (HoFH) individuals from Iberoamerica. Approach and Results: In a cross-sectional retrospective evaluation 134 individuals with a HoFH phenotype, 71 adults (age 39.3±15.8 years, 38.0% males), and 63 children (age 8.8±4.0 years, 50.8% males) were studied. Genetic characterization was available in 129 (96%). The majority (91%) were true homozygotes (true HoFH, n=79, 43.0% children, 46.8% males) or compound heterozygotes (compound heterozygous familial hypercholesterolemia, n=39, 51.3% children, 46.2% males) with putative pathogenic variants in the LDLR. True HoFH due to LDLR variants had higher total (P=0.015) and LDL (low-density lipoprotein)-cholesterol (P=0.008) compared with compound heterozygous familial hypercholesterolemia. Children with true HoFH (n=34) tended to be diagnosed earlier (P=0.051) and had a greater frequency of xanthomas (P=0.016) than those with compound heterozygous familial hypercholesterolemia (n=20). Previous major cardiovascular events were present in 25 (48%) of 52 children (missing information in 2 cases), and in 43 (67%) of 64 adults with LDLR variants. Children who are true HoFH had higher frequency of major cardiovascular events (P=0.02), coronary heart (P=0.013), and aortic/supra-aortic valve diseases (P=0.022) than compound heterozygous familial hypercholesterolemia. In adults, no differences were observed in major cardiovascular events according to type of LDLR variant. From 118 subjects with LDLR variants, 76 (64%) had 2 likely pathogenic or pathogenic variants. In 89 subjects with 2 LDLR variants, those with at least one null allele were younger (P=0.003) and had a greater frequency of major cardiovascular events (P=0.038) occurring at an earlier age (P=0.001). CONCLUSIONS: There was a high frequency of cardiovascular disease even in children. Phenotype and cardiovascular complications were heterogeneous and associated with the type of molecular defect.

Prevalence and risk factors of fatty liver in Portuguese adults.

BACKGROUND: Prevalence of fatty liver (FL) and nonalcoholic fatty liver disease (NAFLD) depends mainly on obesity, diabetes and genetic factors. FL and NAFLD prevalence was evaluated in Portuguese adult population and correlated with several risk factors and related mortality data, within the same period. MATERIALS AND METHODS: A cross-sectional, population-based multicenter study, voluntary and randomly selected in 834 Portuguese adults (18-79 years). Participants were evaluated after 12-hour fasting. Anthropometric data, past history including alcohol consumption, and associated diseases were registered. Blood samples were collected for biochemical testing. Dietary intake was evaluated using a semi-quantitative food frequency questionnaire. Presence of FL was evaluated using ultrasound, and NAFLD was diagnosed after exclusion of other causes for liver disease. RESULTS: Adjusted prevalence of FL and NAFLD was 37.8% and 17.0%, respectively. FL individuals were older, more frequently males, with increased probability of having obesity, diabetes or harmful alcohol consumption (HAC). NAFLD individuals were also older, but had a similar sex distribution and an increased probability of obesity and diabetes. In both groups, no differences were found regarding dietary pattern or physical activity. During the same time period, nonalcoholic steatohepatitis (NASH) liver-related deaths in Portugal were 0.105/100 000, while alcohol-related liver disease mortality was 6.790/100 000. CONCLUSION: The large spectrum of FL was present in more than one third of the population, although only less than half could be classified as NAFLD. Other significant risk factors, such as HAC, are probably implicated in FL, explaining the low NASH-related mortality compared with the high alcohol-related mortality during the same time period.

The familial hypercholesterolaemia phenotype: Monogenic familial hypercholesterolaemia, polygenic hypercholesterolaemia and other causes.

Familial hypercholesterolaemia (FH) is a monogenic disorder characterised by high low-density lipoprotein cholesterol (LDL-C) concentrations and increased cardiovascular risk. However, in clinically defined FH cohorts worldwide, an FH-causing variant is only found in 40%-50% of the cases. The aim of this work was to characterise the genetic cause of the FH phenotype in Portuguese clinical FH patients. Between 1999 and 2017, 731 index patients (311 children and 420 adults) who met the Simon Broome diagnostic criteria had been referred to our laboratory. LDLR, APOB, PCSK9, APOE, LIPA, LDLRAP1, ABCG5/8 genes were analysed by polymerase chain reaction amplification and Sanger sequencing. The 6-SNP LDL-C genetic risk score (GRS) for polygenic hypercholesterolaemia was validated in the Portuguese population and cases with a GRS over the 25th percentile were considered to have a high likelihood of polygenic hypercholesterolaemia. An FH-causing mutation was found in 39% of patients (94% in LDLR, 5% APOB and 1% PCSK9), while at least 29% have polygenic hypercholesterolaemia and 1% have other lipid disorders. A genetic cause for the FH phenotype was found in 503 patients (69%). All known causes of the FH phenotype should be investigated in FH cohorts to ensure accurate diagnosis and appropriate management.

What Is the Role of the New Index Relative Fat Mass (RFM) in the Assessment of Nonalcoholic Fatty Liver Disease (NAFLD)?

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the liver manifestation of adiposopathy. Recently, a new score was developed to estimate body fat percentage (relative fat mass, RFM). We aimed to evaluate the value of RFM in predicting the presence and severity of NAFLD, compared with other anthropometric measurements. METHODS: RFM, body mass index (BMI), and other anthropometric measurements were evaluated in two cohorts of subjects: a cohort from a Portuguese prospective epidemiological study (e_Cor) and morbidly obese patients with biopsy-proven NAFLD. We evaluated if RFM and BMI were related with the presence and severity of liver disease, which was assessed by noninvasive tools in the first cohort and by liver histology in the morbidly obese cohort. The independence of relations found in univariate analysis was assessed with multivariable logistic regression analysis. RESULTS: In the general population cohort, 744 subjects (48% male) were enrolled. BMI-defined obesity was present in 23% and RFM-defined obesity in 86%. Insulin resistance (IR) related with BMI-defined obesity (OR 4.37 [2.16-8.84]) and weight (OR 1.05 [1.02-1.08]) in men, and waist circumference (WC) (OR 1.07 [1.03-1.11]) in women. Dyslipidemia and hypertension related with RFM-defined obesity in men (OR 2.96 [1.36-6.47] and OR 5.37 [1.31-22.06], respectively). Ultrasound-diagnosed NAFLD in 33% related with weight in men (OR 1.03 [1.003-1.06] and WC in women (OR 1.06 [1.02-1.10]). In men, ALT elevation related with weight (OR 1.04 [1.02-1.07]). In women, advanced fibrosis (estimated by NAFLD Fibrosis Score) associated with BMI-defined obesity (OR 42.43 [3.61-498.13]). In the morbidly obese cohort, 152 subjects were enrolled, of whom 84% were female, 37% had steatohepatitis, and 9.4% had advanced fibrosis. Adiponectin associated inversely and leptin positively with RFM in men. The severity of steatosis increased linearly with BMI and WC in women. Higher BMI associated with steatohepatitis in women and advanced fibrosis in men. CONCLUSION: RFM-defined obesity better predicted dyslipidemia and hypertension (though not IR) and adipokine imbalance; however, it did not add value to BMI-defined obesity in predicting NAFLD or liver injury.

No Evidence for Lower Levels of Serum Vitamin D in the Presence of Hepatic Steatosis. A Study on the Portuguese General Population.

Introduction and aims: Nonalcoholic fatty liver disease (NAFLD) has become highly prevalent, paralleling the pandemic of obesity and diabetes, and represents an important burden. Nutrition knowledge is fundamental, in prevention, evolution and treatment of NAFLD. Association of low serum levels of vitamin D (VD) with several diseases, including NAFLD, has been emphasized in the last decade. We evaluated how serum levels of VD correlate with the presence of hepatic steatosis, and VD intake, in a random sample of the Portuguese adult population. Methods: Participants underwent a dietary intake inquiry, using a semi-quantitative food frequency questionnaire representative of the usual intake over the previous year. Anthropometric measures, blood tests and ultrasound were done. Hepatic steatosis was quantified according to Hamaguchi's ultrasonographic score (steatosis defined by a score ≥ 2). Results: We recruited 789 adult individuals, 416 males (52.7%), mean age of 49.9 ± 17.0 years (18-79). Prevalence of hepatic steatosis was 35.5%, and after exclusion of excessive alcohol consumption, 28.0%. Mean VD serum levels were 26.0 ± 9.8 ng/ml and 68.4% participants had serum VD levels below 30 ng/ml. Mean serum levels of VD were not significantly different between participants with steatosis vs. no steatosis: 25.2±8.7 vs. 26.4±10.3 ng/ml, respectively (p=0.071). There was no correlation between VD serum levels and VD intake, measured by the FFQ, r=0.075 (p= 0.383). Conclusions: In spite of a high prevalence rate, there was no evidence that decreased VD serum levels were associated with hepatic steatosis. No significant correlation was found between VD dietary ingestion and VD serum levels.

Further evidence of novel APOB mutations as a cause of familial hypercholesterolaemia.

BACKGROUND AMD AIMS: APOB mutations are a rare cause of familial hypercholesterolaemia (FH) and, until recently, routine genetic diagnosis only included the study of two small APOB fragments. In previous years, 5 novel functional mutations have been described in APOB fragments not routinely studied, our group having functionally characterized 2 of them. The main aim of this work was to identify and characterize novel alterations in APOB to assess the genetic cause of hypercholesterolemia in patients with a clinical diagnosis of FH. METHODS: We performed next generation sequencing of 48 Portuguese clinical FH patients, who were apparently mutation negative. All variants found in APOB were annotated. For functional studies, LDL from index patients and relatives was separated and marked with FITC-LDL for flow cytometry assays in lymphocytes and U937 growth assays. RESULTS: A total of 11 potential pathogenic variants were identified. Variants p.(Pro994Leu) and p.(Thr3826Met) in exons 19 and 26 were found in 4 patients, and in vitro analysis was performed for these variants. An exon 26 alteration (p.(Thr3826Met)) showed a decrease in binding and internalization of LDL, and in U937 growth assays that was similar to the effect with p.(Arg3527Gln). An alteration in exon 19 had a neutral effect. CONCLUSIONS: The spectrum of functional alterations in APOB outside the fragments routinely screened is slowly growing. Screening of all 29 exons of APOB is advised for FH routine diagnosis, but functional characterization is necessary for pathogenicity assessment. It is expected that the number of patients with functional APOB mutations will increase in the near future.

Single-Vesicle Assays Using Liposomes and Cell-Derived Vesicles: From Modeling Complex Membrane Processes to Synthetic Biology and Biomedical Applications.

The plasma membrane is of central importance for defining the closed volume of cells in contradistinction to the extracellular environment. The plasma membrane not only serves as a boundary, but it also mediates the exchange of physical and chemical information between the cell and its environment in order to maintain intra- and intercellular functions. Artificial lipid- and cell-derived membrane vesicles have been used as closed-volume containers, representing the simplest cell model systems to study transmembrane processes and intracellular biochemistry. Classical examples are studies of membrane translocation processes in plasma membrane vesicles and proteoliposomes mediated by transport proteins and ion channels. Liposomes and native membrane vesicles are widely used as model membranes for investigating the binding and bilayer insertion of proteins, the structure and function of membrane proteins, the intramembrane composition and distribution of lipids and proteins, and the intermembrane interactions during exo- and endocytosis. In addition, natural cell-released microvesicles have gained importance for early detection of diseases and for their use as nanoreactors and minimal protocells. Yet, in most studies, ensembles of vesicles have been employed. More recently, new micro- and nanotechnological tools as well as novel developments in both optical and electron microscopy have allowed the isolation and investigation of individual (sub)micrometer-sized vesicles. Such single-vesicle experiments have revealed large heterogeneities in the structure and function of membrane components of single vesicles, which were hidden in ensemble studies. These results have opened enormous possibilities for bioanalysis and biotechnological applications involving unprecedented miniaturization at the nanometer and attoliter range. This review will cover important developments toward single-vesicle analysis and the central discoveries made in this exciting field of research.

Analysis of publicly available LDLR, APOB, and PCSK9 variants associated with familial hypercholesterolemia: application of ACMG guidelines and implications for familial hypercholesterolemia diagnosis.

PurposeFamilial hypercholesterolemia (FH) is an autosomal disorder of lipid metabolism presenting with increased cardiovascular risk. Although more than 1,700 variants have been associated with FH, the great majority have not been functionally proved to affect the low-density lipoprotein receptor cycle. We aimed to classify all described variants associated with FH and to establish the proportion of variants that lack evidence to support their pathogenicity.MethodsWe followed American College of Medical Genetics and Genomics (ACMG) guidelines for the classification, and collected information from a variety of databases and individual reports. A worldwide overview of publicly available FH variants was also performed.ResultsA total of 2,104 unique variants were identified as being associated with FH, but only 166 variants have been proven by complete in vitro functional studies to be causative of disease. Additionally, applying the ACMG guidelines, 1,097 variants were considered pathogenic or likely pathogenic. Only seven variants were found in all five continents.ConclusionThe lack of functional evidence for about 85% of all variants found in FH patients can compromise FH diagnosis and patient prognosis. ACMG classification improves variant interpretation, but functional studies are necessary to understand the effect of about 40% of all variants reported. Nevertheless, ACMG guidelines need to be adapted to FH for a better diagnosis.