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Familial Hypercholesterolemia (FH) is the most common genetic disease caused by variants in LDLR, APOB, PCSK9 genes; it is characterized by high levels of LDL-cholesterol and premature cardiovascular disease. We aim to perform a retrospective analysis of a genetically screened population (528 unrelated patients - 342 adults and 186 children) to evaluate the biochemical and clinical correlations with the different genetic statuses. Genetic screening was performed by traditional sequencing and some patients were re-analyzed by next-generation-sequencing. Pathogenic variants, mainly missense in the LDLR gene, were identified in 402/528 patients (76.1%), including 4 homozygotes, 17 compound heterozygotes and 1 double heterozygotes. A gradual increase of LDL-cholesterol was observed from patients without pathogenic variants to patients with a defective variant, to patients with a null variant and to patients with two variants. Six variants accounted for 51% of patients; a large variability of LDL-cholesterol was observed among patients carrying the same variant. The frequency of pathogenic variants gradually increased from unlikely FH to definite FH, according to the Dutch Lipid Clinic Network criteria. Genetic diagnosis can help prognostic evaluation of FH patients, discriminating between the different genetic statuses or variant types. Clinical suspicion of FH should be considered even if few symptoms are present or if LDL-cholesterol is only mildly increased. This article is protected by copyright. All rights reserved.

Little is known about the role of Lp(a) in the assessment of cardiovascular risk in the paediatric population. Trying to clarify the clinical relevance of Lp(a) in risk stratification, the aim of the study is to evaluate the association between Lp(a) plasma levels in children with familial hypercholesterolaemia (FH) and positive family history for premature cardiovascular disease (pCVD) in first- and second-degree relatives.653 Caucasian children and adolescents (334 females and 319 males), aged 2-17 years, with diagnosis of FH from a paediatric cohort included in the LIPIGEN Network, were selected. We compared family history of pCVD, lipid and genetic profile in two groups based on Lp(a) levels below or above 30 mg/dL. To determine the independent predictors of pCVD, a multivariate logistic regression was used, with all clinical characteristics and blood measurements as predictors.Subjects with Lp(a) 30 mg/dl more frequently reported positive family history of pCVD compared to subjects with Lp(a)≤30 mg/dl (69.90% vs 36.66%, p 0.0001), while did not show differences in terms of median [interquartile range] LDL-cholesterol level (153.00 [88.00 vs 164.50 [90.25] mg/dL, p = 0.3105). In the regression analysis, Lp(a) 30 mg/dl was an independent predictor of family history of pCVD. Comparing subjects with or without family history of pCVD, we reported significant differences for Lp(a) 30 mg/dl (46.25% vs 17.65%, p 0.0001), FH genetic mutation (50.48% vs 40.75%, p = 0,0157), as well as for LDL-cholesterol (p = 0.0013) and total cholesterol (p = 0.0101).Children/adolescents with FH and Lp(a) 30 mg/dl where more likely to have a positive family history of pCVD. Lp(a) screening in children and adolescents with FH may enhance risk assessment and help identify those subjects, children and relatives, at increased pCVD risk.

Background The European Atherosclerosis Society Familial Hypercholesterolaemia Studies Collaboration (FHSC) global registry provides a platform for the global surveillance of familial hypercholesterolaemia through harmonisation and pooling of multinational data. In this study, we aimed to characterise the adult population with heterozygous familial hypercholesterolaemia and described how it is detected and managed globally. Methods Using FHSC global registry data, we did a cross-sectional assessment of adults (aged 18 years or older) with a clinical or genetic diagnosis of probable or definite heterozygous familial hypercholesterolaemia at the time they were entered into the registries. Data were assessed overall and by WHO regions, sex, and index versus non-index cases. Findings Of the 61 612 individuals in the registry, 42 167 adults (21 999 [53.6%] women) from 56 countries were included in the study. Of these, 31 798 (75.4%) were diagnosed with the Dutch Lipid Clinic Network criteria, and 35 490 (84.2%) were from the WHO region of Europe. Median age of participants at entry in the registry was 46.2 years (IQR 34.3-58.0); median age at diagnosis of familial hypercholesterolaemia was 44.4 years (32.5-56.5), with 40.2% of participants younger than 40 years when diagnosed. Prevalence of cardiovascular risk factors increased progressively with age and varied by WHO region. Prevalence of coronary disease was 17.4% (2.1% for stroke and 5.2% for peripheral artery disease), increasing with concentrations of untreated LDL cholesterol, and was about two times lower in women than in men. Among patients receiving lipid-lowering medications, 16 803 (81.1%) were receiving statins and 3691 (21.2%) were on combination therapy, with greater use of more potent lipid-lowering medication in men than in women. Median LDL cholesterol was 5.43 mmol/L (IQR 4.32-6.72) among patients not taking lipid-lowering medications and 4.23 mmol/L (3.20-5.66) among those taking them. Among patients taking lipid-lowering medications, 2.7% had LDL cholesterol lower than 1.8 mmol/L; the use of combination therapy, particularly with three drugs and with proprotein convertase subtilisin-kexin type 9 inhibitors, was associated with a higher proportion and greater odds of having LDL cholesterol lower than 1.8 mmol/L. Compared with index cases, patients who were non-index cases were younger, with lower LDL cholesterol and lower prevalence of cardiovascular risk factors and cardiovascular diseases (all p, Pfizer Independent Grant for Learning Change [16157823]; Amgen; Merck Sharp Dohme; Sanofi-Aventis; Daiichi Sankyo; Regeneron; National Institute for Health Research (NIHR) Imperial Biomedical Research Centre, UK; NIHR; Czech Ministry of Health [NU20-02-00261]; Canadian Institutes of Health Research; Austrian Heart Foundation; Tyrolean Regional Government; Gulf Heart Association, The EAS FHSC is an academic initiative that has received funding from a Pfizer Independent Grant for Learning & Change 2014 (16157823) and from investigator-initiated research grants to the European Atherosclerosis Society-Imperial College London from Amgen, Merck Sharp & Dohme, Sanofi-Aventis, Daiichi Sankyo, and Regeneron. KKR acknowledges support from the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre, UK. KID acknowledges support from a PhD Studentship from NIHR under the Applied Health Research programme for Northwest London, UK (the views expressed in this publication are those of the authors and not necessarily those of the National Health Service, the NIHR, or the Department of Health). TF was supported by a grant from the Czech Ministry of Health (NU20-02-00261). JG receives support from the Canadian Institutes of Health Research. The Austrian Familial Hypercholesterolaemia registry has been supported by funds from the Austrian Heart Foundation and the Tyrolean Regional Government. The Gulf Familial Hypercholesterolaemia registry was done under the auspices of the Gulf Heart Association.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by pathogenic variants in genes encoding for LDL receptor (LDLR), Apolipoprotein B and Proprotein convertase subtilisin/kexin type 9 (PCSK9). Among PCSK9 variants, only Gain-of- Function (GOF) variants lead to FH. Greater attention should be paid to the classification of variants as pathogenic. Two hundred sixty nine patients with a clinical suspect of FH were screened for variants in LDLR and the patients without pathogenic variants were screened for variants in PCSK9 and APOB. Functional characterization of PCSK9 variants was performed by assessment of protein secretion, of LDLR activity in presence of PCSK9 variant proteins as well as of the LDLR affinity of the PCSK9 variants. Among 81 patients without pathogenic variants in LDLR, 7 PCSK9 heterozygotes were found, 4 of whom were carriers of variants whose role in FH pathogenesis is still unknown. Functional characterization revealed that two variants (p.(Ser636Arg) and p.(Arg357Cys)) were GOF variants. In Conclusions, we demonstrated a GOF effect of 2 PCSK9 variants that can be considered as FH-causative variants. The study highlights the important role played by functional characterization in integrating diagnostic procedures when the pathogenicity of new variants has not been previously demonstrated.

Background Familial hypercholesterolemia (FH) is a genetic disorder caused by mutations in genes involved in low-density lipoprotein (LDL) uptake (LDLR, APOB and PCSK9). Genetic diagnosis is particularly useful in asymptomatic children allowing for the detection of definite FH patients. Furthermore, defining their genetic status may be of considerable importance as the compound heterozygous status is much more severe than the heterozygous one. Our study aims at depicting the genetic background of an Italian pediatric population with FH focusing on the correlation between lipid profile and genetic status. Methods Out of 196 patients with clinically suspected FH (LDL-cholesterol [LDL-C] levels above 3.37 mmol/L, cholesterol level above 6.46 mmol/L in a first-degree relative or the presence of premature cardiovascular acute disease in a first/second-degree relative), we screened 164 index cases for mutations in the LDLR, APOB and PCSK9 genes. Results Patients with mutations (129/164) showed increased levels of LDL-C, 95th percentile-adjusted LDL-C and LDL/high-density lipoprotein (HDL) ratio and decreased levels of HDL-C, adjusted HDL-C. The association of the LDL/HDL ratio with the presence of mutations was assessed independently of age, (body mass index) BMI, parental hypercholesterolemia, premature coronary artery disease (CAD), triglycerides by multivariate logistic regression (odds ratio [OR]=1.701 [1.103–2.621], p=0.016). The LDL/HDL ratio gradually increased from patients without mutations to patients with missense mutations, null mutations and compound heterozygotes. Conclusions In conclusion, the LDL/HDL ratio proved to be a better parameter than LDL-C for discriminating patients with from patients without mutations across different genetic statuses.

Children affected by primary hyperlipidemia have a high risk of developing cardiovascular diseases (CVDs) during adulthood. Several studies have reported a positive association between the intake of polyunsaturated fatty acids (PUFAs) and improvements in lipid markers and CVD risk. Dietary supplements may represent a potential strategy in the management of hyperlipidemia. In this context, the effectiveness of hempseed oil (HSO) rich in PUFAs (particularly linoleic acid (LA) and α-linolenic acid (ALA)) in the modulation of hyperlipidemia has been poorly investigated. The present pilot study aimed to explore the impact of HSO supplementation on the serum lipid profile and fatty acid (FA) composition of red blood cells (RBCs) in children and adolescents with primary hyperlipidemia. A randomized, 8 week long, parallel dietary intervention study was performed. Thirty-six hyperlipidemic probands (6-16 years) on diet therapy were randomized into two groups: the HSO group, receiving 3 g of HSO providing 1.4 g of LA and 0.7 g/day of ALA, and the control group. Both groups received specific dietary guidelines. Before and after the intervention, blood samples were collected and the serum lipid profile, FA composition of RBCs and omega-3-index were analyzed. Eight weeks of supplementation with HSO significantly (p .01) reduced the RBC content of total saturated and monounsaturated FAs (-5.02 ± 7.94% and - 2.12 ± 2.23%, respectively), increased the levels of total n-3 and n-6 PUFAs (+1.57 ± 1.96% and + 5.39 ± 7.18%, respectively) and the omega-3 index (+1.18 ± 1.42%), but failed to affect the serum lipid profile compared to the control group. In conclusion, our findings seem to support the contribution of HSO supplementation in improving the RBC phospholipid composition and omega-3 index, while no effect was observed regarding modulation of the lipid profile. Further controlled studies are necessary to achieve a complete understanding of the effects of HSO in the modulation of hyperlipidemia and CVD risk in this and other target groups.

Hyperlipidemia starts at a pediatric age and represents an unquestionable risk factor for cardiovascular disease. Modulation of the intestinal microbial ecosystem (IME), in principle, can ameliorate lipid profiles. In this study, we characterized the IME of children and adolescents with primary hyperlipidemia by analyzing fecal samples through 16S rRNA gene profiling (n = 15) and short chain fatty acid (SCFA) quantification (n = 32). The same analyses were also carried out on age-matched normolipidemic controls (n = 15). Moreover, we evaluated the modulatory effect of regular hazelnut intake (approximately 0.43 g of hazelnuts with skin per kg of body weight) on the IME of 15 children and adolescents with hyperlipidemia for eight weeks. We found alterations of numerous operational taxonomic units potentially associated with SCFA-producing bacteria and reductions in the fecal levels of acetate, butyrate and propionate in hyperlipidemic subjects. Furthermore, we observed that an eight-week hazelnut intervention may induce limited changes in fecal microbiota composition but can significantly modulate the fecal levels of predominant intestinal SCFAs, such as acetate. Finally, correlation analyses indicated that changes in lipidemic parameters are linked to modifications of the abundance of specific bacterial taxa, such as the families Lachnospiraceae and Ruminococcaceae and the genera Akkermansia, Bacteroides, Roseburia, and Faecalibacterium. This study suggests that children and adolescents with primary hyperlipidemia possess an altered IME. The promising results presented here support the need for future dietary interventions aimed at positively modulating the IME of hyperlipidemic subjects.

Objective Primary dyslipidemias are major risk factors for cardiovascular disease and should be addressed early in life. The aim of this study was to evaluate, in children affected by primary hypercholesterolemia, the efficacy and tolerability of a short-term treatment with a dietary supplement containing glucomannan. Methods A double-blind, randomized, placebo-controlled, cross-over trial was conducted in 36 children (aged 6-15 years) affected by primary hypercholesterolemia. After a 4-week run-in period with dietary counseling, children received glucomannan or placebo twice-daily for 8 weeks, separated by a 4-week washout period. Lipid profile was assessed at baseline and after each treatment period. Results Glucomannan significantly reduced total cholesterol (TC) by 5.1% (p = 0.008), low-density lipoprotein cholesterol (LDL-C) levels by 7.3% (p = 0.008) and non-high-density lipoprotein cholesterol by 7.2% (p = 0.002) as compared with placebo. No significant differences were observed in high-density lipoprotein cholesterol, triglyceride, Apolipoprotein B, and Apolipoprotein A-I concentrations. According to sex, glucomannan significantly reduced in females, but not in males, TC (-6.1%, p = 0.011) and LDL cholesterol (-9%, p = 0.015). No major adverse effects were recorded and only few patients experienced transitory intestinal discomfort. Conclusion Treatment with glucomannan of children affected by primary dyslipidemia is well-tolerated and effectively lowers total and LDL cholesterol in females and non-high-density lipoprotein cholesterol, but not Apolipoprotein B in both males and females.

This study aimed at characterizing the fatty acid (FA) composition of red blood cell (RBC) phospholipids in children and adolescents with primary hyperlipidemia, and to ascertain potential association with serum lipid profile and dietary factors. At this purpose, 54 probands aged 6-17 years were recruited. Subjects showed a low omega-3 index (eicosapentaenoic acid, EPA + docosahexaenoic acid, DHA

Summary Background & aim Regular intake of nuts improves lipid profile and thus reduces the cardiovascular (CV) risk associated with hyperlipidemia. The aim of the study was to investigate the effect of a dietary intervention with hazelnuts (HZNs, 15–30 g/day, depending on patient weight) on serum lipid profile, anthropometric parameters and fatty acids (FAs) composition of erythrocyte phospholipids in children and adolescents with primary hyperlipidemia. Methods Eight-week randomized, single blind, controlled, three-arm, parallel-group study. Sixty-six subjects were enrolled and randomized in 3 groups receiving: 1) hazelnuts with skin (HZN+S); 2) hazelnuts without skin (HZN-S); 3) dietary advices for hyperlipidemia only (controls). Before and after intervention, clinical parameters were measured and blood samples were collected for the evaluation of serum lipid levels and phospholipid FA composition of erythrocytes. Results Two-way ANOVA showed a significant effect of time on serum low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)/LDL-C ratio and non-HDL-C (p ≤ 0.001), but not of treatment and time × treatment interaction. In particular, HZN+S and HZN-S significantly reduced the concentrations of LDL-C and increased HDL-C/LDL-C ratio. HZNs also had a favorable impact on FAs composition of erythrocyte phospholipids, as demonstrated by time × treatment interaction, with a significant increase of monounsaturated fatty acids (MUFAs) (p = 0.008) and MUFAs/saturated fatty acids (SFAs) ratio (p = 0.002) with respect to the control group. Conclusions For the first time, we documented a positive effect of HZN consumption on lipid profile and FA composition of erythrocyte phospholipids in children with primary hyperlipidemia. Further studies are encouraged to better define HZN impact on the markers of CV risk in this population. The trial was registered under ISRCTN.com , ID no. ISRCTN12261900.

Purpose: The purpose of this study was to enhance understanding of lysosomal acid lipase deficiency (LALD) in infancy. Genet Med 18 5, 452–458. Methods: Investigators reviewed medical records of infants with LALD and summarized data for the overall population and for patients with and without early growth failure (GF). Kaplan–Meier survival analyses were conducted for the overall population and for treated and untreated patients. Genet Med 18 5, 452–458. Results: Records for 35 patients, 26 with early GF, were analyzed. Prominent symptom manifestations included vomiting, diarrhea, and steatorrhea. Median age at death was 3.7 months; estimated probability of survival past age 12 months was 0.114 (95% confidence interval (CI): 0.009-0.220). Among patients with early GF, median age at death was 3.5 months; estimated probability of survival past age 12 months was 0.038 (95% CI: 0.000-0.112). Treated patients (hematopoietic stem cell transplant (HSCT), n = 9; HSCT and liver transplant, n = 1) in the overall population and the early GF subset survived longer than untreated patients, but survival was still poor (median age at death, 8.6 months). Genet Med 18 5, 452–458. Conclusions: These data confirm and expand earlier insights on the progression and course of LALD presenting in infancy. Despite variations in the nature, onset, and severity of clinical manifestations, and treatment attempts, clinical outcome was poor. Genet Med 18 5, 452–458.

Objective To investigate in children and adolescents with familial dyslipidemias the association between lipoprotein(a) [Lp(a)] level and family history of cardiovascular disease (CVD), and whether this association is independent of the disturbed lipid profile. Study design Lp(a) level, lipid profile, and a 2-generation genealogic tree to detect cardiovascular events were evaluated in 231 patients with familial dyslipidemias. Lp(a) levels were stratified according to presence, age of occurrence, and number and type of cardiovascular events in the patient's kindreds. Results Lp(a) and other plasma lipid fractions did not differ between patients with and those without a family history of cardiovascular events. However, the percentage of patients with elevated Lp(a) level (≥85th percentile) was higher in those with a positive family history for early cardiovascular events ( P = .01). Lp(a) level was a significant independent predictor of the number of premature cardiovascular events (β = 0.17; P = .01) and of cerebrovascular events in kindreds (OR, 2.5; 95% CI, 1.05-6.03; P = .039), independent of plasma lipid fractions and other cardiovascular risk factors. Conclusions In children and adolescents with familial dyslipidemias, the overall association between Lp(a) level and family history of early CVD may be due to a threshold effect in those with the highest Lp(a) levels. However, multiple cardiovascular events and cerebrovascular events are predicted by any increase in plasma Lp(a) level, independent of other cardiovascular risk factors.

Serum lipoproteins influence cell cholesterol content by delivering and removing cholesterol to/from cells, functions mainly exerted by LDL and HDL, respectively. Especially in the case of HDL, structure and composition are crucial for function, beyond serum levels. Cholesteryl ester storage disease (CESD) is caused by LIPA gene mutations and reduced activity of lysosomal acid lipase (LAL), the enzyme responsible for hydrolysis of cholesteryl esters and TG. CESD patients typically present dyslipidaemia, liver damage and premature atherosclerosis. The objective of this work was to evaluate serum HDL cholesterol efflux capacity (CEC) and serum cholesterol loading capacity (CLC) in CESD pediatric patients and to study lipoprotein qualitative modifications.HDL CEC was evaluated by radioisotopic techniques, serum CLC was measured by a fluorimetric assay, HDL subclasses were determined by two-dimensional electrophoresis.CESD patients (n = 3) displayed on average increased LDL cholesterol (+163%; p = 0.019), TG (+203; p = 0.012), phospholipids (+40%; p = 0.024) and lower HDL cholesterol (-57%; p = 0.012) compared to controls (n = 9). CESD HDL CEC was impaired both as a whole (average reduction of 26%; p0.0001) and with respect to specific membrane cholesterol transporters (-23% for aqueous diffusion; p = 0.005; -32% for ABCA1-efflux; p = 0.0002; -60% for SR-BI-efflux; p0.0001; -42% for ABCG1-efflux p = 0.0003). A marked reduction in the pre-β HDL concentration (-69%; p = 0.012) was detected. Finally, CESD serum CLC was significantly increased (+21%; p = 0.0007).These new data demonstrate that the pro-atherogenic modifications of serum include disturbances in lipoprotein functions involved in cell cholesterol homeostasis occurring from very early age in CESD patients.

Objective Preclinical investigations support the use of probiotics in the treatment of hypercholesterolemia, but clinical evidence is often contrasting. The aim of this study was to evaluate the effects of a probiotic formulation containing three Bifidobacterium strains on lipid profiles in children affected by primary dyslipidemia. Methods Thirty-eight children with dyslipidemia, ages 10.8 ± 2.1 y, were enrolled in a randomized, double-blind, placebo-controlled cross-over study. After a 4-wk diet run-in period, the children received probiotics ( B. animalis subspecies lactis MB 2409, B. bifidum MB 109B, and B. longum subspecies longum BL04) or placebo for 3 mo. After 1 mo, wash-out treatments were switched. A strict dietary evaluation concerning satured fatty acids and cholesterol content, STEP I diet accordingly, was performed by a dietitian who examined the weekly dietary diary at each visit. Results Baseline lipid profile was (mean ± SD): total cholesterol (TC) 222.8 ± 23.2 mg/dL, high-density lipoprotein cholesterol (HDL-C) 55.8 ± 12.2 mg/dL, triglycerides (TG) 99.0 ± 61.7 mg/dL, and low-density lipoprotein cholesterol (LDL-C) 147.2 ± 21.9 mg/dL. After 3 mo of probiotic treatment, the lipid profile was: TC 211.9 ± 27.3 mg/dL, HDL-C 60.7 ± 14.2 mg/dL, TG 79.5 ± 34.5 mg/dL, and LDL-C 135.3 ± 24.2 mg/dL. Compared with placebo, probiotics reduced TC by 3.4% ( P = 0.02) and LDL-C by 3.8% ( P = 0.001). No significant dietary change occurred through the study and no relevant adverse effects were observed. Conclusions Treatment with a Bifidobacterium probiotic formulation was well tolerated and useful in combination with to diet therapy. Children with dyslipidemia benefited from this approach, although the results need to be confirmed by larger controlled studies.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Plant sterols lower serum cholesterol concentration. Available data have confirmed the lipid-lowering efficacy in adults, while there is a relative dearth of data in children and almost exclusively restricted to subjects with familial hypercholesterolemia (FH). Aim of the present study was to evaluate the efficacy, tolerability and safety of plant sterol supplementation in children with different forms of primary hyperlipidemias. The effect of plant sterol consumption on plasma lipids was evaluated in 32 children with heterozygous FH, 13 children with Familial Combined Hyperlipidemia (FCH) and 13 children with Undefined Hypercholesterolemia (UH) in a 12-week open-label intervention study using plant sterol-enriched yoghurt. Plasma lipids and apolipoproteins were measured by routine methods. Markers of cholesterol synthesis (lathosterol) and absorption (campesterol and sitosterol) were measured by GC-MS. Tolerability and adherence to recommended regimen was very high. A significant reduction was observed in LDL-cholesterol in the three groups (10.7, 14.2 and 16.0% in FH, FCH and UH, respectively). Lathosterol concentrations were unchanged, reflecting a lack of increased synthesis of cholesterol. Of the two absorption markers, only sitosterol showed a slight but significant increase. Daily consumption of plant sterol dairy products favorably changes lipid profile by reducing LDL-cholesterol. To our knowledge, this is the first report of the use of plant sterols-enriched foods in treating children with primary hyperlipidemia such as FCH and UH, likely to be the most frequent form also in the young age in the western populations.

We describe a new fully reliable method for the differential diagnosis of tetrahydrobiopterin-dependent hyperphenylalaninaemia (HPA). The method comprises the combined phenylalanine (Phe) plus tetrahydrobiopterin (BH4) oral loading test and enables the selective screening of BH4 deficiency when pterin analysis is not available or when a clear diagnosis has not been previously made. It should be performed together with the measurement of dihydropteridine reductase (DHPR) activity in blood. The new combined loading test was performed in nine patients with primary HPA, three with classical phenylketonuria (PKU), three with DHPR deficiency, and three with 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. Three hours after oral Phe loading (100 mg/kg body weight), synthetic BH4 was administered orally at doses of either 7.5 or 20 mg/kg body weight. Amino acid (Phe and tyrosine) and pterin (neopterin and biopterin) metabolism and kinetics were analysed. By exploiting the decrease in serum Phe 4 and 8 h after administration, a clear response was obtained with the higher BH4 dose (20 mg/kg body weight), allowing detection of all cases of BH4 deficiency, as well as differentiation of BH4 synthesis from regeneration defects. Since DHPR deficient patients who were previously shown to be non-responsive to the simple BH4 loading test gave a positive response, the combined Phe plus BH4 loading test can be used as a more reliable tool for the differential diagnosis of HPA in these patients. Moreover, it takes advantage of being performed while patients are on a Phe-restricted diet.

The relationship between blood phenylalanine concentrations and serum and erythrocyte biopterin and neopterin concentrations was investigated in 20 phenylketonuric patients with different dietary compliance. At serum phenylalanine concentrations ranging from 43 to 1004 μmol/l, a good correlation was found with serum biopterin (r = 0.76, P < 0.001) and with red blood cell biopterin (r = 0.62, P < 0.001). A similar correlation was found between serum neopterin and phenylalanine (r = 0.60, P < 0.001). The correlation between red blood cell neopterin and serum phenylalanine was less evident, however (r = 0.47, P < 0.005). After oral loading with phenylalanine (100 mg/kg body weight), serum and red blood cell biopterin concentrations increased in patients with classical phenylketonuria as well as in one patient with dihydropteridine reductase deficiency in response to the induced acute hyperphenylalaninemia. One patient suffering from 6-pyruvoyl tetrahydropterin synthase deficiency was loaded orally with tetrahydrobiopterin (20 mg/kg body weight). The kinetics of administered cofactor confirmed its rapid absorption, with early increase of serum concentrations followed by its transport into the red blood cells. The half-life of biopterin was approximately 7 h in serum and 15 h in red blood cells. Because both values are less than the half-life of phenylalanine (20–30 h) in serum, biopterin measurement offers no advantage in monitoring dietary control in hyperphenylalaninemic patients.

It is now widely accepted that tetrahydrobiopterin (BH4), the natural cofactor of aromatic amino acid hydroxylases, in the absence of its regenerating enzyme dihydropteridine reductase (DHPR), will function only stoichiometrically in the phenylalanine (Phe) hydroxylating system. This has limited the use of pterin cofactor in diagnosis and treatment of patients suffering from inherited DHPR deficiency, one of the most common forms of hyperphenylalaninemia caused by BH4 deficiency. This is despite the observation of a dramatic fall in serum Phe concentration after BH4 loading in such patients. In this study, quantitation of this phenomenon was obtained by comparing the kinetics of serum Phe after either a simple Phe or a combined Phe plus BH4 oral loading in patients with Phe hydroxylase or with DHPR deficiency. Only in the latter was the total body clearance of Phe enhanced up to 5 times by the cofactor administration, resulting in the molar equivalent of Phe hydroxylated/mol of BH4 ranging from at least 6 to 10, against the postulated 1. As a consequence, BH4 administration should be attempted therapeutically in DHPR-deficient patients, thus avoiding a lifelong Phe-restricted diet. Preliminary experience with such treatment is given with two cases.

The prevention of cardiovascular risk, as occurs in lipoprotein disorders, is required since childhood. Aim of the study was to evaluate, in a group of children affected by primary dyslipidemia, the efficacy, tolerability and safety of a short-term treatment with a dietary supplement containing red yeast rice extract and policosanols.40 children affected by heterozygous Familial Hypercholesterolemia (FH) (n=24) and Familial Combined Hyperlipidemia (FCH) (n=16), aged 8-16 years, were enrolled in a double-blind, randomized, placebo-controlled, cross-over trial. After a 4-week run-in period with only dietary advice, children received a dietary supplement containing 200mg red yeast rice extract, corresponding to 3mg of monacolins, and 10mg policosanols once-daily and placebo for 8 weeks, separated by a 4-week washout period. Lipid profile was assessed after each treatment period. The dietary supplement, compared with the placebo, significantly reduced total cholesterol by 18.5% (p0.001), LDL-C levels by 25.1% (p0.001), and apolipoprotein B by 25.3% (p0.001) when patients were considered as a whole group. Similar results were obtained when FH and FCH were considered separately and no significant difference between groups was detected. No significant differences were observed in HDL-C and apolipoprotein A-I levels. No adverse effects were detected when liver and muscular enzymes (AST, ALT, and CK) were determined.The treatment with a dietary supplement containing red yeast rice extract and policosanols has been for the first time successfully employed in hypercholesterolemic children. Results indicate this strategy as an effective, safe and well tolerated in a short-term trial.

Objective To ascertain whether the molecular characterization of a defect in the low-density lipoprotein (LDL) receptor gene ( LDLR ) in children with heterozygous familial hypercholesterolemia (heFH) identifies subjects at greater risk of developing premature coronary artery disease (pCAD) later in life. Study design We investigated 264 children with heFH from 201 families, along with 148 affected parents and 100 unaffected siblings. The lipid profile was assessed before any treatment was provided, and genotype analysis was performed to characterize LDLR defects. In a subgroup of children with heFH and controls, we measured aorta and carotid intima-media thickness (aIMT and cIMT). The prevalence of pCAD in parents and/or grandparents with heFH was recorded. Results The children with heFH with a family history of pCAD had higher LDL cholesterol and apolipoprotein B levels and greater aIMT and cIMT than those with negative family history. Compared with carriers of LDLR -defective mutations, carriers of LDLR -negative mutations had a more severe phenotype, in terms of plasma lipid levels and IMT, and a higher prevalence of pCAD in first-degree relatives (36% vs 6.7%; P Conclusions The study of heFH in children, in which other risk factors for CAD play a minor role, allows early identification of those at increased risk for developing pCAD, who merit more stringent clinical control and early pharmacologic treatment.

Prospective studies demonstrated an increased cardiovascular risk in subjects with high levels of either the endothelial-platelet activation marker P-selectin or high-sensitivity C-reactive protein (hs-CRP). Both children with heterozygous familial hypercholesterolemia (FH) and those with familial combined hyperlipidemia (FCHL) are prone to premature atherosclerosis. Our objective was to investigate in children with either FH or FCHL whether P-selectin and hs-CRP contribute to carotid intima-media thickness (IMT), along with increased plasma lipid levels.Carotid IMT, serum lipids and soluble P-selectin and hs-CRP levels were measured in 88 children (mean age 10.5+/-4.3 years) including 44 dyslipidemic children (25 with FH and 19 with FCHL) and 44 non-dyslipidemic controls.Carotid IMT was significantly higher among dyslipidemic than in control children (0.46+/-0.06mm vs 0.43+/-0.06mm, p=0.003) and serum P-selectin levels as well [129(50-254)ng/mL vs 50(24.5-130)ng/mL, p0.001]. FH but not FCHL children had higher hs-CRP levels than controls [0.7(0.01-6.9)mg/L vs 0.3(0.1-1.2)mg/L, p=0.006]. In the entire sample of dyslipidemic children, carotid IMT was positively associated with soluble P-selectin levels (rho=0.30, p=0.049), but not with hs-CRP. The association between P-selectin and carotid IMT was independent from confounders, including plasma lipid levels.Endothelial-platelet activation, more than low-grade systemic inflammation, correlates with premature atherosclerosis among children with familial dyslipidemia, this association being independent from plasma lipid levels.

Objectives— The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African-Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. Methods and Results— We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest (th ) percentile, 44 in the highest (>95 th ) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delG) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. Conclusions— We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals.

BACKGROUND: Sitosterolaemia is a rare autosomal recessive disorder characterised by elevated plasma levels of plant sterols and cholesterol. Sitosterolaemia is caused by gene mutations in either of two ATP-binding cassette (ABC) half transporters, ABCG5 and ABCG8. The plasma sterol profile and genetic analysis of a 10-year-old girl who had tuberous xanthomas is the subject of this report. MATERIALS AND METHODS: Genomic DNA was isolated from white blood cells from the proband, her family and a control group of healthy people. All exons of ABCG5 and ABCG8 were sequenced. Plasma cholesterol and triglycerides were measured by routine methods. All other plasma sterols were measured by Gas Chromatography coupled to Mass Spectrometry. RESULTS: The proband was found to be homozygous for a single nucleotide mutation in exon 10 of the ABCG5 gene, consisting of a C to T transition at nucleotide 1336 of the coding sequence, which results in the premature termination of the ABCG5 protein at amino acid 446 (Arg446X). Her mother and brother were also homozygous for the same mutation and all had elevated plasma beta-sitosterol levels. The father was heterozygous and showed normal beta-sitosterol levels. This mutation was not found in healthy normolipidaemic subjects. CONCLUSIONS: We describe a novel nonsense mutation in exon 10 of the ABCG5 gene in a 10-year-old girl showing clinical and biochemical features of sitosterolaemia. This family study broadens the spectrum of the ABCG5/ABCG8 mutations causing sitosterolaemia and helps highlight the correlations between such gene mutations, biochemical phenotype and the development of cardiovascular disease.

Prenatal diagnosis of tetrahydrobiopterin (BH4) deficiency was undertaken by evaluating the pterin patterns in amniotic fluid and the specific enzyme activities in fetal or extrafetal tissues. This allowed the prenatal diagnosis in 19 pregnancies at risk. In 8 families with a child already affected by dihydropteridine reductase deficiency 4 fetuses were diagnosed as homozygotes and 4 as heterozygotes for the defect. In 11 families with a child affected by 6-pyruvoyl tetrahydropterin synthase deficiency 4 fetuses were homozygous, 4 heterozygous and 3 normal. This study also advanced our knowledge of tetrahydrobiopterin metabolism during fetal development. The key enzymes involved in the biosynthesis of BH4 are expressed early and allow the fetus to be autotrophous for its cofactor requirement. In a twin pregnancy, both fetuses were diagnosed to be heterozygotes for dihydropteridine reductase deficiency and primapterin (7-biopterin) in amniotic fluid was increased. This indicates that pterin-4 alpha-carbinolamine dehydratase activity seems to be differently expressed during fetal life. As a consequence, pterins detected in amniotic fluid are of fetal origin and 6- and 7-substituted pterins can be present in amniotic fluid in higher proportions when compared with other body fluids.

Maple syrup urine disease (MSUD) is an autosomal recessive disease due to deficiency of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) caused by a large number of mutations. In the present study, DNA from Italian patients and their relatives was examined for three point mutations (Y393N in the E(1 alpha) gene, T841G and G1031A in the E(2) gene) and two deletions (-G at the intron/exon border of exon 8 in the E(2) gene and an 11bp deletion in exon 1 of the E(1 beta) gene) using the polymerase chain reaction (PCR) followed by allele-specific oligonucleotide (ASO) hybridization, gene-scanning size analysis of fluorescent-tagged PCR products and/or automated DNA sequence analysis. Our results show that two different mutations account for 7 of the 20 mutant MSUD alleles, Two unrelated affected children, two of their parents and one sibling were carriers for the 11bp deletion in the E(1 beta) gene, one patient and her mother were heterozygous for Y393N in E(1 alpha,) while T841G, G1031A and the -G deletion in E(2) were not detected. This study is the first attempt to characterize at a nucleic acid level MSUD mutations in Italy. Our results indicate that additional defects are present in the Italian population and that, unlike the Mennonites, a number of different MSUD mutations exist in Italians.

Some cases of primary hyperphenylalaninemia are not caused by the lack of phenylalanine hydroxylase, but by the lack of its cofactor tetrahydrobiopterin. These patients are not clinically responsive to a phenylalanine-restricted diet, but need specific substitution therapy. Thus, it became necessary to examine all newborns screened as positive with the Guthrie test for tetrahydrobiopterin deficiency. Methods based on urinary pterin or on specific enzyme activity measurements are limited in their availability, and the simplest method, based on the lowering of serum phenylalanine after loading with cofactor, was discouraged by the finding that some dihydropteridine reductase-deficient patients were unresponsive. The preliminary observation that this limitation could be overcome by increasing the dose of the administered cofactor prompted us to reevaluate the potential of the tetrahydrobiopterin loading test in hyperphenylalaninemia. Fifteen patients, eight with ultimate diagnosis of phenylketonuria, three with 6-pyruvoyl tetrahydropterin synthase-, and four with dihydropteridine reductase-deficiency, have been examined by administering synthetic tetrahydrobiopterin both orally, at doses of 7.5 and 20 mg/kg, and i.v., at a dose of 2 mg/kg. All the tetrahydrobiopterin-deficient patients, unlike those with phenylketonuria, responded to the oral dose of 20 mg/kg cofactor by lowering their serum phenylalanine concentration markedly below baseline to an extent easily detectable by Guthrie cards. This method allows for a simple screening method when enzyme or pterin studies are not available.

Atypical phenylketonuria (PKU) is a group of very rare and severe diseases caused by tetrahydrobiopterin (BH4) deficiency (Niederwieser and Curtius, 1987). So far three inborn errors of metabolism are known to cause BH4 deficiency. defects in: GTP cyclohydrolase I (GTPCH); 6-pyruvoyl tetrahydropterin synthase (PPH4S); and dihydropteridine reductase (DHPR) (Blau, 1988). Recently a new form of atypical PKU with unusual 7-iso-biopterin excretion in the urine of patients was described (Curtius et al., 1988). Prenatal diagnosis of BH4 deficiency can be achieved mainly by measurement of pterin metabolites in amniotic fluid and of enzyme activities in cultured fluid cells and fetal blood (Blau et al., 1987).

Amniocentesis was performed at 19 weeks gestation in a mother who had previously delivered a boy with "dihydrobiopterin synthetase" (DHBS) deficiency. The amniotic fluid contained neopterin in high (136 nmol/l) and biopterin in very low concentrations (1.8 nmol/l). The activity of the phosphate-eliminating enzyme (PEE, also called 6-pyruvoyl tetrahydropterin synthase, substrate: 7,8-dihydroneopterin triphosphate) which is present in liver and erythrocytes and defective in DHBS deficiency, was measured in the erythrocytes of the family members. The fetal sample showed only 2% of the activity of healthy adult controls and was comparable with that of the affected sibling. Obligate heterozygotes had activities around 20% of the controls. Two fetal control samples showed even higher activities than adult erythrocytes, Sepiapterin reductase activities wer normal in all cases. At autopsy, PEE deficiency was confirmed in the liver of the fetus. We concluded that DHBS deficiency (and most probably also GTP cyclohydrolase I deficiency) can be diagnosed by metabolite measurements in amniotic fluid. PEE activity is measurable in erythrocytes, although the assay needs to be improved. Since maternal tetrahydrobiopterin does not cross the placenta, treatment of a tetrahydrobiopterin-deficient fetus with tetrahydrobiopterin in utero is not possible.

Correlation of the response to a load of tetrahydrobiopterin (BH4) in dihydropterin reductase (DHPR) deficient patients to the type of mutation in these patients has led to the conclusion that 4 patients without mutant DHPR molecules in their cells respond to the BH4 load, whereas 3 patients with mutant DHPR in their cells do not respond. Intravenous injection of BH4 in 1 of the cases not responding to BH4 again showed no response.

Two patients with dihydropteridine reductase (DHPR) deficiency, in one case due to the absence of any enzyme protein (DHPR- cross reactive material (CRM)-) and in the other case due to the production of a mutant type devoid of catalytic activity (DHPR- CRM+) were examined. This latter form of malignant phenylketonuria, whose relative frequency seems to be higher in the Italian population, possibly has a worse prognosis. The earlier onset and the greater severity of clinical symptoms are associated with a more pronounced hydroxylation defect, as shown by higher degree of neonatal hyperphenylalaninaemia, unresponsiveness to an oral tetrahydrobiopterin load, lower concentrations of neurotransmitter metabolites, and reduced tyrosine production after an oral phenylalanine load.

Background A significant proportion of individuals clinically diagnosed with familial hypercholesterolemia (FH), but without any disease-causing mutation, are likely to have polygenic hypercholesterolemia. We evaluated the distribution of a polygenic risk score, consisting of 12 low-density lipoprotein cholesterol (LDL-C)-raising variants (polygenic LDL-C risk score), in subjects with a clinical diagnosis of FH. Methods and Results Within the Lipid Transport Disorders Italian Genetic Network (LIPIGEN) study, 875 patients who were FH-mutation positive (women, 54.75%; mean age, 42.47±15.00 years) and 644 patients who were FH-mutation negative (women, 54.21%; mean age, 49.73±13.54 years) were evaluated. Patients who were FH-mutation negative had lower mean levels of pretreatment LDL-C than patients who were FH-mutation positive (217.14±55.49 versus 270.52±68.59 mg/dL, P