Long-term Benefits and Harms Associated With Genetic Cholesteryl Ester Transfer Protein Deficiency in the General Population.

Importance: The balance between the potential long-term clinical benefits and harms associated with genetic cholesteryl ester transfer protein (CETP) deficiency, mimicking pharmacologic CETP inhibition, is unknown. Objective: To assess the relative benefits and harms associated with genetic CETP deficiency. Design, Setting, and Participants: This study examined 2 similar prospective cohorts of the Danish general population, with data on a total of 102 607 participants collected from October 10, 1991, through December 7, 2018. Exposures: Weighted CETP allele scores. Main Outcomes and Measures: Incident cardiovascular mortality, ischemic heart disease, myocardial infarction, ischemic stroke, peripheral arterial disease, vascular dementia, Alzheimer disease, all-cause mortality, and age-related macular degeneration (AMD). The study first tested whether a CETP allele score was associated with morbidity and mortality, when scaled to genetically lower levels of non-high-density lipoprotein (HDL) cholesterol (ie, 17 mg/dL), corresponding to the reduction observed for anacetrapib vs placebo in the Randomized Evaluation of the Effects of Anacetrapib Through Lipid-Modification (REVEAL) trial. Second, the study assessed how much of the change in morbidity and mortality was associated with genetically lower levels of non-HDL cholesterol. Finally, the balance between the potential long-term clinical benefits and harms associated with genetic CETP deficiency was quantified. For AMD, the analyses also included higher levels of HDL cholesterol associated with genetic CETP deficiency. Results: Of 102 607 individuals in the study, 56 559 (55%) were women (median age, 58 years [IQR, 47-67 years]). Multivariable adjusted hazard ratios showed that a genetically lower level of non-HDL cholesterol (ie, 17 mg/dL) was associated with a lower risk of cardiovascular mortality (hazard ratio [HR], 0.77 [95% CI, 0.62-0.95]), ischemic heart disease (HR, 0.80 [95% CI, 0.68-0.95]), myocardial infarction (HR, 0.72 [95% CI, 0.55-0.93]), peripheral arterial disease (HR, 0.80 [95% CI, 0.63-1.02]), and vascular dementia (HR, 0.38 [95% CI, 0.18-0.80]) and an increased risk of AMD (HR, 2.33 [95% CI, 1.63-3.30]) but was not associated with all-cause mortality (HR, 0.91 [95% CI, 0.81-1.02]), ischemic stroke (HR, 1.05 [95% CI, 0.81-1.36]), or Alzheimer disease (HR, 1.25 [95% CI, 0.89-1.76]). When scaled to a higher level of HDL cholesterol, the increased risk of AMD was even larger. A considerable fraction of the lower risk of cardiovascular end points was associated with genetically lower levels of non-HDL cholesterol, while the higher risk of AMD was associated with genetically higher levels of HDL cholesterol. Per 1 million person-years, the projected 1916 more AMD events associated with genetically higher levels of HDL cholesterol was similar to the 1962 fewer events of cardiovascular mortality and myocardial infarction combined associated with genetically lower levels of non-HDL cholesterol. Conclusions and Relevance: This study suggests that genetic CETP deficiency, mimicking pharmacologic CETP inhibition, was associated with a lower risk of cardiovascular morbidity and mortality, but with a markedly higher risk of AMD.

Novel mutations associated with carnitine-acylcarnitine translocase and carnitine palmitoyl transferase 2 deficiencies in Malaysia.

OBJECTIVE: Carnitine-acylcarnitine Translocase (CACT) deficiency (OMIM 212138) and carnitine palmitoyl transferase 2 (CPT2) deficiency (OMIM 60065050) are rare inherited disorders of mitochondrial long chain fatty acid oxidation. The aim of our study is to review the clinical, biochemical and molecular characteristics in children diagnosed with CACT and CPT2 deficiencies in Malaysia. DESIGN AND METHODS: This is a retrospective study. We reviewed medical records of six patients diagnosed with CACT and CPT2 deficiencies. They were identified from a selective high-risk screening of 50,579 patients from January 2010 until Jun 2020. RESULTS: All six patients had either elevation of the long chain acylcarnitines and/or an elevated (C16 + C18:1)/C2 acylcarnitine ratio. SLC25A20 gene sequencing of patient 1 and 6 showed a homozygous splice site mutation at c.199-10 T > G in intron 2. Two novel mutations at c.109C > T p. (Arg37*) in exon 2 and at c.706C > T p. (Arg236*) in exon 7 of SLC25A20 gene were found in patient 2. Patient 3 and 4 (siblings) exhibited a compound heterozygous mutation at c.638A > G p. (Asp213Gly) and novel mutation c.1073 T > G p. (Leu358Arg) in exon 4 of CPT2 gene. A significant combined prevalence at 0.01% of CACT and CPT2 deficiencies was found in the symptomatic Malaysian patients. CONCLUSIONS: The use of the (C16 + C18:1)/C2 acylcarnitine ratio in dried blood spot in our experience improves the diagnostic specificity for CACT/CPT2 deficiencies over long chain acylcarnitine (C16 and C18:1) alone. DNA sequencing for both genes aids in confirming the diagnosis.

ANGPTL3 and ANGPTL8 in Thai subjects with hyperalphalipoproteinemia and severe hypertriglyceridemia.

BACKGROUND: The role of ANGPTL3 and ANGPTL8 in lipid regulation in patients with very high levels of HDL-cholesterol and triglyceride is unknown. OBJECTIVE: We examined plasma levels of ANGPTL3 and ANGPTL8 in subjects with hyperalphalipoproteinemia (HALP) and in those with severe hypertriglyceridemia (HTG). METHODS: Plasma ANGPTL3 and ANGPTL8 levels were measured by ELISA in 320 subjects, consisting of HALP subjects with HDL-cholesterol ≥100 mg/dl (n=90) and healthy controls (n=90) and subjects with triglyceride ≥886 mg/dl (n=89) and control subjects (n=51). RESULTS: The mean plasma ANGPTL3 level was significantly higher in the HALP group compared to that of the controls (297 ± 112 ng/mL vs. 230 ± 100 ng/mL, p<0.001). Similarly, the mean plasma ANGPTL8 level was also higher in the HALP group (30 ± 11 ng/mL vs. 20 ± 8 ng/mL, p<0.001). Both ANGPTL3 and ANGPTL8 levels positively correlated with HDL-cholesterol levels. In the severe HTG group, plasma ANGPTL3 level was significantly higher than those in the control group (223 ± 105 ng/mL vs. 151 ± 60 ng/mL, p<0.001), but not ANGPTL8 (23 ± 20 ng/mL vs. 31 ± 23 ng/mL in controls, p=0.028). Only ANGPTL3, but not ANGPTL8, levels positively correlated with triglyceride levels. CONCLUSION: Plasma level of ANGPTL3 was increased in both HALP and severe HTG whereas an increase in plasma level of ANGPTL8 was found only in HALP, and not in severe HTG, suggesting that both ANGPTL3 and ANGPTL8 might play distinct roles in lipid regulation on these two extremes of dyslipidemia.

Genetic basis and hematologic manifestations of sitosterolemia in a group of Turkish patients.

BACKGROUND: Sitosterolemia is a rare lipid disorder caused by mutations in adenosine triphosphate-binding cassette genes (ABCG) 5 and 8. OBJECTIVE: To evaluate the phenotypic/genotypic features of sitosterolemia in a group of Turkish patients. METHODS: Seven probands with unexplained hematologic abnormalities and their 13 relatives were enrolled. Sterol levels were measured by gas chromatography and genetic studies were performed using Sanger sequencing. Individuals were diagnosed with sitosterolemia if they were found to have frankly elevated sitosterol level >15 µg/mL and/or pathogenic variants of the ABCG5/ABCG8. RESULTS: The seven probands and their six relatives  were diagnosed with frank sitosterolemia, and all these patients had hematologic abnormalities. The remaining seven relatives were asymptomatic heterozygous carriers. Three novel variants in the ABCG5 gene (c.161G>A, c.1375C>T, IVS10-1G>T), one novel variant in the ABCG8 gene (c.1762G>C) and one known variant in the ABCG5 gene (c.1336 C>T) were identified. No variant was identified in one case. The mean sitosterol level was significantly higher and mean platelet count was significantly lower in patients with homozygous variants compared to heterozygous variants (p<0.05, for all). Diets low in plant sterols were recommended for 13 symptomatic cases. Four homozygotes received ezetimibe, and their splenomegaly, anemia, and thrombocytopenia completely resolved except one. CONCLUSION: The five pathogenic variants identified in this study indicate the genetic heterogeneity of sitosterolemia in Turkish population. Patients with unexplained hematologic abnormalities (specifically macrothrombocytopenia) should have their sterol level measured as initial testing. Ezetimibe can be a good choice for sitosterolemia.

Quantitation and evaluation of perinatal medium-chain and long-chain acylcarnitine blood concentrations in 12,000 full-term breastfed newborns.

OBJECTIVES: Medium-chain (MCA) and long-chain acylcarnitine (LCA) blood concentrations play a significant role in the fatty acid (FA) oxidation process, especially during the first days of life. Identification of their abnormal concentrations, via expanded newborn screening, can lead to the diagnosis of FA oxidation disorders. This study aimed to demonstrate MCA and LCA concentrations in Dried Blood Spots (DBS) of full-term breastfed infants, in relation to their birth weight (BW) perinatally. METHODS: Breastfed full-term infants (n = 12,000, 6,000 males, 6,000 females) with BW 2,000-3,999 g were divided into four equal groups: Group A, 2,000-2,499 g, B 2,500-2,999 g, C 3,000-3,499 g, and D 3,500-3,999 g. Samples were collected as DBS and acylcarnitines were determined via a liquid chromatography tandem mass spectrometry method. RESULTS: MCA and LCA blood concentrations were determined significantly lower in group A (low birth weight infants) in both sexes. Infants with BW > 3,500 g (group D), were characterized by lower levels of C10, C10:1, C14, C14:1 acylcarnitines and higher levels of C16 and C18:1 acylcarnitines, as compared to the other groups of this study. CONCLUSIONS: Concentration patterns in full-term breastfed newborns in relation to sex and mainly BW found in this study could be very helpful for neonatologists, especially for newborns of group A.

Unexpected elevation in valproic acid concentration and agranulocytosis in a patient with short-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an autosomal recessive metabolic disorder or condition of fatty acid ß-oxidation, caused by mutations in the gene encoding SCAD (ACADS). We report an infant with SCAD deficiency who unexpectedly exhibited an extremely high blood concentration of valproic acid (VPA) and agranulocytosis. CASE REPORT: An 8-month-old girl was diagnosed with West syndrome (infantile spasms), and VPA was administered at the standard level of 25 mg/kg/day. However, the blood concentration of VPA rose unexpectedly to 230 µg/mL (two- to three-fold higher than the expected value), and continued to remain relatively high even after the dosage was reduced (7 mg/kg/day, blood concentration of 88 µg/mL). Furthermore, she presented with a high-grade fever with agranulocytosis (neutrophil 231/µL). The abnormal pharmacokinetics and toxicity of VPA raised the suspicion of possible inborn errors of metabolism in the fatty acid ß-oxidation pathway. Blood tandem mass spectrometry revealed a transient elevation of C4, and urine gas chromatography-mass spectrometry revealed a continuous elevation of ethylmalonate. Finally, gene analysis revealed compound heterozygous mutations, c.625G > A (p.G209S) and c.1031A > G (p.E344G), in ACADS. CONCLUSION: VPA should be avoided if a patient is suspected to have inborn errors of ß-oxidation including SCAD deficiency.

Effects of fasting, feeding and exercise on plasma acylcarnitines among subjects with CPT2D, VLCADD and LCHADD/TFPD.

BACKGROUND: The plasma acylcarnitine profile is frequently used as a biochemical assessment for follow-up in diagnosed patients with fatty acid oxidation disorders (FAODs). Disease specific acylcarnitine species are elevated during metabolic decompensation but there is clinical and biochemical heterogeneity among patients and limited data on the utility of an acylcarnitine profile for routine clinical monitoring. METHODS: We evaluated plasma acylcarnitine profiles from 30 diagnosed patients with long-chain FAODs (carnitine palmitoyltransferase-2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD), and long-chain 3-hydroxy acyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCHAD/TFP) deficiencies) collected after an overnight fast, after feeding a controlled low-fat diet, and before and after moderate exercise. Our purpose was to describe the variability in this biomarker and how various physiologic states effect the acylcarnitine concentrations in circulation. RESULTS: Disease specific acylcarnitine species were higher after an overnight fast and decreased by approximately 60% two hours after a controlled breakfast meal. Moderate-intensity exercise increased the acylcarnitine species but it varied by diagnosis. When analyzed for a genotype/phenotype correlation, the presence of the common LCHADD mutation (c.1528G > C) was associated with higher levels of 3-hydroxyacylcarnitines than in patients with other mutations. CONCLUSIONS: We found that feeding consistently suppressed and that moderate intensity exercise increased disease specific acylcarnitine species, but the response to exercise was highly variable across subjects and diagnoses. The clinical utility of routine plasma acylcarnitine analysis for outpatient treatment monitoring remains questionable; however, if acylcarnitine profiles are measured in the clinical setting, standardized procedures are required for sample collection to be of value.

Institutional experience of newborn screening for inborn metabolism disorders by tandem MS in the Turkish population.

Background The tandem mass spectrometry method in the screening of congenital metabolic disorders is not included in routine national newborn screening programmes in Turkey. To evaluate the distribution of acylcarnitines and amino acid levels in normal newborns, establish acylcarnitine and amino acid cut-off levels and further preliminary results of inherited metabolic disorders inferentially in the Turkish population. Methods Newborn screening tests performed by tandem MS from 2016 to 2018 were retrospectively reviewed. The study group included 17,066 newborns born in our hospitals located in various regions of Turkey. Blood samples were obtained from infants older than 24 h of age. Among the 17,066 newborns, the metabolic screening data of 9,994 full-term newborns (>37 weeks) were employed to obtain the percentile distribution of the normal population. The study group (17,066) was screened for 26 types of inborn error of metabolism. Results Our established cut-offs, were compared with the cut-offs determined by Region for Stork Study and Centers for Disease Control. Among the 26 screened disorders, a total of 12 cases (8 amino acid metabolism disorders, 1 urea cycle defect, 2 organic acidaemias and 1 fatty acid oxidation disorder) were identified. Conclusions Because of the high rate of consanguineous marriages in Turkey, the development of a nationwide screening panel is necessary for early detection and management of potentially treatable inherited metabolic disorders.

Ethnic variability in newborn metabolic screening markers associated with false-positive outcomes.

Newborn screening (NBS) programmes utilise information on a variety of clinical variables such as gestational age, sex, and birth weight to reduce false-positive screens for inborn metabolic disorders. Here we study the influence of ethnicity on metabolic marker levels in a diverse newborn population. NBS data from screen-negative singleton babies (n = 100 000) were analysed, which included blood metabolic markers measured by tandem mass spectrometry and ethnicity status reported by the parents. Metabolic marker levels were compared between major ethnic groups (Asian, Black, Hispanic, White) using effect size analysis, which controlled for group size differences and influence from clinical variables. Marker level differences found between ethnic groups were correlated to NBS data from 2532 false-positive cases for four metabolic diseases: glutaric acidemia type 1 (GA-1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). In the result, 79% of the metabolic markers (34 of 43) had ethnicity-related differences. Compared to the other groups, Black infants had elevated GA-1 markers (C5DC, Cohen's d = .37, P < .001), Hispanics had elevated MMA markers (C3, Cohen's d = .13, P < .001, and C3/C2, Cohen's d = .27, P < .001); and Whites had elevated VLCADD markers (C14, Cohen's d = .28, P < .001, and C14:1, Cohen's d = .22, P < .001) and decreased OTCD markers (citrulline, Cohen's d = -.26, P < .001). These findings correlated with the higher false-positive rates in Black infants for GA-1, in Hispanics for MMA, and in Whites for OTCD and for VLCADD. Web-based tools are available to analyse ethnicity-related changes in newborn metabolism and to support developing methods to identify false-positives in metabolic screening.

Features of Sitosterolemia in Children.

Sitosterolemia is a rare lipid metabolism disease with heterogeneous manifestations. Atherosclerosis can occur in children, and therefore, early detection, diagnosis, and treatment of this disease are important. We studied 18 pediatric patients with sitosterolemia who showed a significant increase in plasma lipid levels and analyzed their clinical, biochemical, and genetic characteristics. We recorded the initial serum lipid results and clinical manifestations of the patients. Lipid and plant sterol levels were measured after homozygous or compound heterozygous mutations of ABCG5 or ABCG8 were identified by genetic testing. Plasma plant sterol levels were analyzed by gas chromatography. Fourteen cases of sitosterolemia were examined by ultrasound and echocardiography. The initial total cholesterol and low-density lipoprotein levels of the children were significantly increased, but then markedly decreased after diet control or drug treatment, and even reached normal levels. Carotid atherosclerosis and aortic valve regurgitation were present in three of 14 patients. Serum lipid levels of children with sitosterolemia and xanthomas were notably higher than those without xanthomas. There were no significant differences in clinical manifestations between patients with different genotypes. In conclusion, sitosterolemia should be considered in children with hyperlipidemia who do not present with xanthomas, especially with a significant increase in total cholesterol and low-density lipoprotein levels. There does not appear to be a correlation between clinical phenotype and genotype.

Prediction of VLCAD deficiency phenotype by a metabolic fingerprint in newborn screening bloodspots.

PURPOSE: Newborns who test positive for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in newborn screening may have a severe phenotype with early onset of life-threatening symptoms but may also have an attenuated phenotype and never become symptomatic. The objective of this study is to investigate whether metabolomic profiles in dried bloodspots (DBS) of newborns allow early phenotypic prediction, permitting tailored treatment and follow-up. METHODS: A metabolic fingerprint was generated by direct infusion high resolution mass spectrometry in DBS of VLCADD patients (n = 15) and matched controls. Multivariate analysis of the metabolomic profiles was applied to differentiate subgroups. RESULTS: Concentration of six acylcarnitine species differed significantly between patients and controls. The concentration of C18:2- and C20:0-carnitine, 13,14-dihydroretinol and deoxycytidine monophosphate allowed separation between mild and severe patients. Two patients who could not be prognosticated on early clinical symptoms, were correctly fitted for severity in the score plot based on the untargeted metabolomics. CONCLUSION: Distinctive metabolomic profiles in DBS of newborns with VLCADD may allow phenotypic prognostication. The full potential of this approach as well as the underlying biochemical mechanisms need further investigation.

MiRNAs as biomarkers of phenotype in neutral lipid storage disease with myopathy.

BACKGROUND: Neutral lipid storage disease with myopathy (NLSDM) is a rare lipid metabolism disorder. In this study, we evaluated some circulating miRNAs levels in serum samples and the MRI of three affected siblings. METHODS: Three members of one NLSDM family were identified: two brothers and one sister. Muscles of lower and right upper extremities were studied by MRI. Expression profile of miRNAs, obtained from serum samples, was detected using qRT-PCR. RESULTS: Two brothers presented with progressive skeletal myopathy, while the sister had severe hepatosteatosis and diabetes. NLSDM patients showed a significant increase of muscle-specific miRNAs expression compared with healthy subjects. We found a correlation between hepatic damage and elevation of miRNAs expression profile of liver origin. CONCLUSIONS: The dysregulation of miRNAs might represent an indicator of skeletal and hepatic damage and it might be useful to monitor the progression of NLSDM.

Phytosterolemia and γ-glutamyl transferase in adults with parenteral nutrition: Fish versus vegetal lipids: A randomized clinical trial.

OBJECTIVES: Fish oil (FO)-based lipid emulsions (LEs) have been reported to prevent hepatic dysfunction in patients treated with parenteral nutrition (PN). We studied patients with alterations of γ-glutamyl transferase (GGT) associated with the administration of PN containing olive/soybean (O/S)-based LE. The aim of this study was to determine whether the strategy of reducing the lipid dose by 50%, by changing to an FO-based LE, reduced plasma levels of phytosterols (PS) and GGT more effectively and safely, than the strategy of reducing lipid contribution by 50% while maintaining the same LE composition. METHODS: A randomized double-blind clinical trial was carried out in patients with normal initial GGT, who after a minimum of 1 wk of daily PN (0.8 g/kg of O/S-based LE) presented with GGT values twice the upper normal value. At the time of randomization 1:1, lipids were reduced to 0.4 g/kg daily. Group A maintained O/S LE and group B changed to FO LE. The primary endpoints were reduction of plasmatic PS and GGT on day 7 after randomization, performed in the study population per protocol by Student's t test and simple linear regression. Secondary outcomes included alkaline phosphatase (AP), alanine transaminase (ALT), and total bilirubin (BIL), and safety variables. RESULTS: Nineteen patients were included. On day 7 after randomization, GGT and AP values were higher in the O/S group (n = 10; GGT: median [Med], 4.99; interquartile range [IQR], 4.09; AP: Med, 2.59 µkat/L; IQR 1.74) than in the FO group (n = 9; GGT: Med, 2.26 µkat/L; IQR, 1.07; AP: Med, 1.2 µkat/L; IQR 1.44). Although there were no differences in ALT and BIL values, the ALT decrease was larger and more statistically significant in the FO group than in the O/S group (P = 0.009). Total PS (Med, 21.10 µg/mL; IQR, 5.50) in the O/S group was higher than in the FO group (Med, 13.4 µg/mL; IQR, 10.65; P = 0.002). Significant decreases in PS and their fractions were observed, with the exception of campesterol and stigmasterol. CONCLUSION: Plasma accumulation of PS and high values of GGT, AP, and ALT can be prevented with the exclusive administration of FO-based LE.

Changes in pediatric plasma acylcarnitines upon fasting for refined interpretation of metabolic stress.

BACKGROUND: Childhood fasting intolerance is a life-threatening problem associated with various inborn errors of metabolism. Plasma acylcarnitines reflect fatty acid oxidation and help determine fasting intolerance etiology. Pediatric reference values of plasma acylcarnitines upon fasting are not available, complicating interpretation of stress samples. METHODS: Retrospective analysis of supervised clinical fasting studies between 01/2005-09/2012. Exclusion criteria involved patients with (suspected) disorders, repeated tests or incomplete results. Remaining children were grouped according to age: group A (≤24 months), B (25-84 months) and C (≥85 months). Median and 2.5th to 97.5th percentiles of basic metabolic parameters and acylcarnitines were determined at start and end of testing on the ward and analyzed for significant differences (p<0.05). RESULTS: Out of 127 fasting studies, 48 were included: group A (n=13), B (n=23) and C (n=12). Hypoglycemia occurred in 21%. Children from group C demonstrated significantly higher end glucose concentrations while end ketone body concentrations were significantly lower compared to younger children. In all groups, free carnitine and C3-carnitine significantly decreased upon fasting, while C2-, C6-, C12:1-, C12-, C14:1-, C14-, C16:1- and C16-carnitine significantly increased. End concentrations of C6-, C12:1-, C12-, C14:1-, C14-, C16:1-, C16- and C18:1-carnitine were significantly lower in children ≥85 months compared to younger children. CONCLUSIONS: Fasting-induced counter-regulatory mechanisms to maintain energy homeostasis are age-dependent. This influences the changes in basic metabolic parameters and acylcarnitine profiles. Our data enable improved interpretation of the individual fasting response and may support assessment of minimal safe fasting times or treatment responses in patients.