Prevalence and predictive role of hypertriglyceridemia in statin-treated patients at very high risk: Insights from the START study.

BACKGROUND AND AIM: Elevated triglyceride (TG) levels seem to identify subjects at increased cardiovascular risk, independent of LDL-C levels. We sought to evaluate the predictive role of hypertriglyceridemia, defined as TG levels ≥150 mg/dl, in very high risk (VHR) patients with chronic coronary syndromes (CCS) treated with statins. METHODS AND RESULTS: Using the data from the STable Coronary Artery Diseases RegisTry (START) study, an Italian nationwide registry, we assessed the association between the TG levels and baseline clinical characteristics, pharmacological treatment and major adverse cardio-cerebrovascular events (MACCE) at 1 year in a large cohort of statin-treated patients at VHR. Of the 4751 consecutive patients with CCS enrolled in the registry and classified as VHR, 2652 (55.8%) had TG values available (mean 120.6 ± 54.9) and were treated with at least a statin at baseline: 2019 (76.1%) with TG < 150 and 633 (23.9%) with TG ≥ 150 mg/dl. At 1 year from enrolment, MACCE occurred in 168 (6.3%) patients, without differences between the two groups of TG (5.9 vs 7.6%; p = 0.14). At multivariable analysis, hypertriglyceridemia did not result as independent predictor of the MACCE (hazard ratio: 1.16; 95% confidence intervals: 0.82-1.64; p = 0.42). CONCLUSIONS: In the present large, nationwide cohort of consecutive CCS patients at VHR with statin-controlled LDL-C levels, hypertriglyceridemia was present in around 24% of cases and did not result as predictor of MACCE at 1 year. Further studies with a longer follow-up and larger sample size are needed to better define the prognostic role of TG levels when intensive LDL lowering therapies are used.

Comparison of two polygenic risk scores to identify non-monogenic primary hypocholesterolemias in a large cohort of Italian hypocholesterolemic subjects.

BACKGROUND: Primary Hypobetalipoproteinemias (HBL) are a group of dominant and recessive monogenic genetic disorders caused by mutations in APOB, PCSK9, ANGPTL3, MTTP, Sar1b genes and characterized by plasma levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB) below the 5th percentile of the distribution in a given population. Mutations in the candidate genes account only for a small proportion of subjects with HBL suggesting a role for a polygenic contribution to the low cholesterol phenotype. OBJECTIVE: To explore the complex genetic architecture of HBL we compared two polygenic risk scores in order to assess the role of the polygenic burden and the differences in the clinical phenotype between monogenic and polygenic HBL; we studied a cohort of 170 subjects with primary HBL referred over a 25-year period to 2 Italian reference centers have been studied. METHODS: The genetic analyses have been based on: Sanger sequencing, in-house NGS customized panel and two scores, PRS1 and PRS2 for the polygenic burden. RESULTS: Sixty 60 (35%) and 63 (37%) subjects had a monogenic and polygenic HBL respectively. LDL-C plasma levels were significantly lower in monogenic HBL (30.87 ± 3.12 mg/dl) compared with the non-monogenic HBL (42.80 ± 2.18 mg/dl) (p<0.002) with no differences in the percentage of fatty liver. CONCLUSION: Only PRS1 is effective in detecting polygenic HBL while PRS2 does not improve the polygenic diagnosis.

rs629301 CELSR2 polymorphism confers a ten-year equivalent risk of critical stenosis assessed by coronary angiography.

BACKGROUND AND AIMS: Novel genetic determinants associated with coronary artery disease (CAD) have been discovered by genome wide association studies. Variants encompassing the CELSR2- PSRC1-SORT1 gene cluster have been associated with CAD. This study is aimed to investigate the rs629301 polymorphism association with the extent of CAD evaluated by coronary angiography (CAG), and to evaluate its associations with an extensive panel of lipid and lipoprotein measurements in a large Italian cohort of 2429 patients. METHODS AND RESULTS: The patients were collected by four Intensive Care Units located in Palermo and Verona (Italy). Clinical Records were filed, blood samples were collected, lipids and apolipoproteins (apo) were measured in separate laboratories. CAD was defined by the presence of stenotic arteries (>50% lumen diameter) by CAG. The presence of CAD was associated with the rs629301 genotype. Patients with CAD were 78% and 73% (p = 0.007) of the T/T vs. T/G + G/G genotype carriers respectively. T/T genotype was also correlated with the number of stenotic arteries, with a 1.29 (1.04-1.61) risk to have a three-arteries disease. T/T genotype correlated with higher levels of LDL-, non-HDL cholesterol, apoB, apoE and apoCIII, and lower HDL-cholesterol. Logistic Regression confirmed that rs629301was associated with CAD independently from the common risk factors, with a risk similar to that conferred by ten years of age [odds ratios were 1.43 (1.04-1.96) and 1.39 (1.22-1.58) respectively]. CONCLUSIONS: rs629301 risk allele was independently associated with the extension and severity of CAD and positively with apoE and apoB containing lipoproteins.

Lack of phenotypic additive effect of familial defective apolipoprotein B3531 in familial hypercholesterolaemia.

Familial defective apolipoprotein (apo) B (FDB) and familial hypercholesterolaemia (FH) are the two common genetic conditions that cause hypercholesterolaemia. R3531C mutation of the APOB gene is a rare cause of FDB. Individuals with both FDB and FH are rare. A 51-year-old man with hypercholesterolaemia (11.4 mmol/L) and his family were studied. Low-density lipoprotein (LDL) receptor (LDLR) and APOB genes were analysed by direct sequencing. LDL of four subjects were studied in a fibroblast LDL receptor-binding displacement assay. We found a mutation of the LDLR gene (p.Y398X) in the proband and in four other family members: the p.R3531C APOB gene mutation was also found in the proband, his father and his children. The proband and his son were thus compound heterozygotes for both FH and FDB. Double heterozygotes did not show higher cholesterol levels compared to carriers of LDLR gene mutation alone. LDL from one of the carriers of the p.R3531C alone exhibited a binding ability, which was similar to a normal subject. This is the first report in Italy of the p.R3531C mutation, and our results show that this mutation has no effect in LDLR p.Y398X/APOB p.R3531C double heterozygotes.

DeepSRE: Identification of sterol responsive elements and nuclear transcription factors Y proximity in human DNA by Convolutional Neural Network analysis.

SREBP1 and 2, are cholesterol sensors able to modulate cholesterol-related gene expression responses. SREBPs binding sites are characterized by the presence of multiple target sequences as SRE, NFY and SP1, that can be arranged differently in different genes, so that it is not easy to identify the binding site on the basis of direct DNA sequence analysis. This paper presents a complete workflow based on a one-dimensional Convolutional Neural Network (CNN) model able to detect putative SREBPs binding sites irrespective of target elements arrangements. The strategy is based on the recognition of SRE linked (less than 250 bp) to NFY sequences according to chromosomal localization derived from TF Immunoprecipitation (TF ChIP) experiments. The CNN is trained with several 100 bp sequences containing both SRE and NF-Y. Once trained, the model is used to predict the presence of SRE-NFY in the first 500 bp of all the known gene promoters. Finally, genes are grouped according to biological process and the processes enriched in genes containing SRE-NFY in their promoters are analyzed in details. This workflow allowed to identify biological processes enriched in SRE containing genes not directly linked to cholesterol metabolism and possible novel DNA patterns able to fill in for missing classical SRE sequences.

PCSK9-D374Y mediated LDL-R degradation can be functionally inhibited by EGF-A and truncated EGF-A peptides: An in vitro study.

BACKGROUND AND AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) through the LDLR epidermal growth factor-like repeat A (EGF-A) domain and induces receptor internalization and degradation. PCSK9 has emerged as a novel therapeutic target for hypercholesterolemia. Clinical studies with PCSK9 inhibiting antibodies have demonstrated strong LDL-c lowering effects, but other therapeutic approaches using small molecule inhibitors for targeting PCSK9 functions may offer supplementary therapeutic options. The aim of our study was to evaluate the effect of synthetic EGF-A analogs on mutated (D374Y) PCSK9-D374Y mediated LDLR degradation in vitro. METHODS: Huh7 human hepatoma cells were transiently transfected to overexpress the gain-of-function D374Y PCSK9 mutation, which has been associated with severe hypercholesterolemia in humans. RESULTS: Transient transfection of cells with PCSK9-D374Y expression vector very effectively enhanced degradation of mature LDLR in Huh7. Treatment with both EGF-A and EGF-A truncated peptides inhibited this effect and showed increased LDLR protein in Huh7 cells transfected with PCSK9-D374Y in a clear concentration dependent manner. Huh7 transfected cells treated with increasing concentration of EGF-A analogs also showed an increase internalization of labeled Dil-LDL. CONCLUSIONS: The result of our study shows that EGF-A analogs are able to effectively hamper the enhanced degradation of LDLR in liver cells expressing PCSK9-D374Y.

Therapeutic Options for Homozygous Familial Hypercholesterolemia: The Role of Lomitapide.

BACKGROUND: Lomitapide (Juxtapid® in US and Lojuxta® in Europe) is the first developed inhibitor of the Microsomal Triglyceride Transfer Protein (MTP) approved as a novel drug for the management of Homozygous Familial Hypercholesterolemia (HoFH). It acts by binding directly and selectively to MTP thus decreasing the assembly and secretion of the apo-B containing lipoproteins both in the liver and in the intestine. AIMS: The present review aims at summarizing the recent knowledge on lomitapide in the management of HoFH. RESULTS: The efficacy and safety of lomitapide have been evaluated in several trials and it has been shown a reduction of the plasma levels of Low-Density Lipoprotein Cholesterol (LDL-C) by an average of more than 50%. Although the most common side effects are gastrointestinal and liver events, lomitapide presents generally with a good tolerability and satisfactory patients compliance. Recently, in Europe, to evaluate the long-term safety and efficacy of lomitapide, the LOWER registry (ClinicalTrials.gov Identifier: NCT02135705) has been established in order to acquire informations on HoFH lomitapidetreated patients from "real life" clinical practice. Furthermore, the observation that lomitapide decreases triglyceride levels may be considered for patients affected by severe forms of hypertriglyceridemia who undergo recurrent episodes of pancreatitis and are poor responders to conventional treatment. CONCLUSION: Lomitapide represents an innovative and efficacious drug for the treatment of HoFH. Longterm safety data, treatment of pediatric and pregnant HoFH patients and management of severe hypertriglyceridemia still require further investigations.

Anti-PCSK9 treatment: is ultra-low low-density lipoprotein cholesterol always good?

Anti-PCSK9 (proprotein convertase subtilisin kexin 9) monoclonal antibodies (Mab) are novel, potent lipid-lowering drugs. They demonstrated to improve the lipid profile in high cardiovascular risk patients. Anti-PCSK9 Mab inhibit the targeted low-density lipoprotein (LDL)-receptor degradation induced by PCSK9 protein and are able to reduce LDL cholesterol (LDL-C) levels on top of conventional lipid-lowering therapy. Though these drugs proved to be very safe in the short-term, little is known about the possible long-term effects, due to the short period of their marketing. The genetic low cholesterol syndromes (LCS) represent the natural models of the lipid-lowering anti-PCSK9 therapy, and a valuable opportunity to predict the long-term effects of these drugs. By looking at the clinical features of such models, we could be able to foresee possible drug-induced side effects. In the present review, the correspondences and discordances between the side effects of anti-PCSK9 therapy and the corresponding LCS models will be examined in the attempt to forecast possible long-term consequences of these novel lipid-lowering agents.

Genetic epidemiology of autosomal recessive hypercholesterolemia in Sicily: Identification by next-generation sequencing of a new kindred.

BACKGROUND: Autosomal recessive hypercholesterolemia (ARH) is a rare inherited lipid disorder. In Sardinia, differently from other world regions, the mutated allele frequency is high. It is caused by mutations in the low-density lipoprotein receptor adaptor protein 1 gene. Fourteen different mutations have been reported so far; in Sardinia, 2 alleles (ARH1 and ARH2) explain most of the cases. Four ARH patients, all carriers of the ARH1 mutation, have been identified in mainland Italy and 2 in Sicily. OBJECTIVE: The objectives of the study were to improve the molecular diagnosis of familial hypercholesterolemia (FH) and to estimate the frequency of the ARH1 allele in 2 free-living Sicilian populations. METHODS: We sequenced by targeted next-generation sequencing 20 genes related to low-density lipoprotein metabolism in 50 hypercholesterolemic subjects. Subjects from 2 free-living populations from Northern (Ventimiglia Heart Study, 848 individuals) and Southern Sicily (Zabut Zabùt Aging Project, 1717 individuals) were genotyped for ARH1 allele. RESULTS: We identified 1 homozygous carrier of the ARH1 mutation among the 50 hypercholesterolemic outpatients. Population-based genotyping of ARH1 in 2565 subjects allowed the identification of 1 heterozygous carrier. The overall estimated allele frequency of ARH1 in Sicily was 0.0002 (0.02%). CONCLUSIONS: The identification of a new case of ARH in Sicily among 50 clinically diagnosed FH highlights the importance of next-generation sequencing analysis as tool to improve the FH diagnosis. Our results also indicate that ARH1 carrier status is present in ∼1:2500 of Sicilian inhabitants, confirming that ARH is extremely rare outside Sardinia.

Threshold Effects of Circulating Angiopoietin-Like 3 Levels on Plasma Lipoproteins.

Context: Angiopoietin-like 3 (ANGPTL3) deficiency in plasma due to loss-of-function gene mutations results in familial combined hypobetalipoproteinemia type 2 (FHBL2) in homozygotes. However, the lipid phenotype in heterozygotes is much milder and does not appear to relate directly to ANGPTL3 levels. Furthermore, the low-density lipoprotein (LDL) phenotype in carriers of ANGPTL3 mutations is unexplained. Objective: To determine whether reduction below a critical threshold in plasma ANGPTL3 levels is a determinant of lipoprotein metabolism in FHBL2, and to determine whether proprotein convertase subtilisin kexin type 9 (PCSK9) is involved in determining low LDL levels in this condition. Design: We studied subjects from 19 families with ANGPTL3 mutations and subjects with familial combined hypobetalipoproteinemia type 1 (FHBL1) due to truncated apolipoprotein B (apoB) species. Results: First, total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and HDL and LDL particle concentration correlated with plasma ANGPTL3 levels but only when the latter was <25% of normal (<60 ng/dL). Second, the very low-density lipoprotein particle concentration correlated strongly with plasma ANGPTL3 when the latter was <58% of normal. Third, both FHBL1 and FHBL2 subjects showed low levels of mature and LDL-bound PCSK9 and higher levels of its furin-cleaved form. Finally, LDL-bound PCSK9 is protected from cleavage by furin and binds to the LDL receptor more strongly than apoB-free PCSK9. Conclusions: Our results suggest that the hypolipidemic effects of ANGPTL3 mutations in FHBL2 are dependent on a threshold of plasma ANGPTL3 levels, with differential effects on various lipoprotein particles. The increased inactivation of PCSK9 by furin in FHBL1 and FHBL2 is likely to cause increased LDL clearance and suggests novel therapeutic avenues.

Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

BACKGROUND: Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. OBJECTIVE: The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. METHODS: We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. RESULTS: The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. CONCLUSIONS: We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG.

The role of registries in rare genetic lipid disorders: Review and introduction of the first global registry in lipoprotein lipase deficiency.

A good understanding of the natural history of rare genetic lipid disorders is a pre-requisite for successful patient management. Disease registries have been helpful in this regard. Lipoprotein Lipase Deficiency (LPLD) is a rare, autosomal-recessive lipid disorder characterized by severe hypertriglyceridemia and a very high risk for recurrent acute pancreatitis, however, only limited data are available on its natural course. Alipogene tiparvovec (Glybera®) is the first gene therapy to receive Marketing Authorization in the European Union; GENIALL (GENetherapy In the MAnagement of Lipoprotein Lipase Deficiency), a 15-year registry focusing on LPLD was launched in 2014 as part of its Risk Management Plan. The aim of this publication is to introduce the GENIALL Registry within a structured literature review of registries in rare genetic lipid disorders. A total of 11 relevant initiatives/registries were identified (homozygous Familial Hypercholesterolemia (hoFH) [n = 5]; LPLD [n = 1]; Lysosomal Acid Lipase Deficiency [LALD, n = 1], detection of mutations in genetic lipid disorders [n = 4]). Besides one product registry in hoFH and the LALD registry, all other initiatives are local or country-specific. GENIALL is the first global prospective registry in LPLD that will collect physician and patient generated data on the natural course of LPLD, as well as long-term outcomes of gene therapy. CONCLUSION: There is a limited number of international initiatives focusing on the natural course of specific rare genetic lipid disorders. The GENIALL LPLD Registry could be the first step towards a future broader global initiative that collects data related to familial chylomicronemia syndrome and their underlying genetic causes.

Association between familial hypobetalipoproteinemia and the risk of diabetes. Is this the other side of the cholesterol-diabetes connection? A systematic review of literature.

Statin therapy is beneficial in reducing LDL cholesterol (LDL-C) levels and cardiovascular events, but it is associated with the risk of incident diabetes mellitus (DM). Familial hypercholesterolemia (FH) is characterized by genetically determined high levels of plasma LDL-C and a low prevalence of DM. LDL-C levels seem then inversely correlated with prevalence of DM. Familial hypobetalipoproteinemia (FHBL) represents the genetic mirror of FH in terms of LDL-C levels, very low in subjects carrying mutations of APOB, PCSK9 (FHBL1) or ANGPTL3 (FHBL2). This review explores the hypothesis that FHBL might represent also the genetic mirror of FH in terms of prevalence of DM and that it is expected to be increased in FHBL in comparison with the general population. A systematic review of published literature on FHBL was made by searching PubMed (1980-2016) for articles presenting clinical data on FHBL probands and relatives. The standardized prevalence rates of DM in FHBL1 were similar to those of the reference population, with a prevalence rate of 8.2 and 9.2%, respectively, while FHBL2 showed a 4.9% prevalence of DM. In conclusion, low LDL-C levels of FHBL do not seem connected to DM as it happens in subjects undergoing statin therapy and the diabetogenic effect of statins has to be explained by mechanisms that do not rely exclusively on the reduced levels of LDL-C. The review also summarizes the published data on the effects of FHBL on insulin sensitivity and the relationships between FH, statin therapy, FHBL1 and intracellular cholesterol metabolism, evaluating possible diabetogenic pathways.

Myristic acid is associated to low plasma HDL cholesterol levels in a Mediterranean population and increases HDL catabolism by enhancing HDL particles trapping to cell surface proteoglycans in a liver hepatoma cell model.

BACKGROUND: HDL-C plasma levels are modulated by dietary fatty acid (FA), but studies investigating dietary supplementation in FA gave contrasting results. Saturated FA increased HDL-C levels only in some studies. Mono-unsaturated FA exerted a slight effect while poly-unsaturated FA mostly increased plasma HDL-C. AIMS: This study presents two aims: i) to investigate the relationship between HDL-C levels and plasma FA composition in a Sicilian population following a "Mediterranean diet", ii) to investigate if FA that resulted correlated with plasma HDL-C levels in the population study and/or very abundant in the plasma were able to affect HDL catabolism in an "in vitro" model of cultured hepatoma cells (HepG2). RESULTS: plasma HDL-C levels in the population correlated negatively with myristic acid (C14:0, ß = -0.24, p < 0.01), oleic acid (C18:1n9, ß = -0.22, p < 0.01) and cis-11-Eicosenoic (C20:1n9, ß = -0.19, p = 0.01) and positively with palmitoleic acid (C16:1, ß = +0.19, p = 0.03). HepG2 cells were conditioned with FA before evaluating HDL binding kinetics, and only C14:0 increased HDL binding by a non-saturable pathway. After removal of heparan sulphate proteoglycans (HSPG) by heparinases HDL binding dropped by 29% only in C14:0 conditioned cells (p < 0.05). C14:0 showed also the highest internalization of HDL-derived cholesteryl esters (CE, +32% p = 0.01 vs. non-conditioned cells). CONCLUSIONS: C14:0 was correlated with decreased plasma HDL-C levels in a Mediterranean population. C14:0 might reduce HDL-C levels by increasing HDL trapping to cell surface HSPG and CE stripping from bound HDL. Other mechanisms are to be investigated to explain the effects of other FA on HDL metabolism.

FragClust and TestClust, two informatics tools for chemical structure hierarchical clustering analysis applied to lipidomics. The example of Alzheimer's disease.

Lipidomic analysis is able to measure simultaneously thousands of compounds belonging to a few lipid classes. In each lipid class, compounds differ only by the acyl radical, ranging between C10:0 (capric acid) and C24:0 (lignoceric acid). Although some metabolites have a peculiar pathological role, more often compounds belonging to a single lipid class exert the same biological effect. Here, we present a lipidomics workflow that extracts the tandem mass spectrometry data from individual files and uses them to group compounds into structurally homogeneous clusters by chemical structure hierarchical clustering analysis (CHCA). The case-to-control peak area ratios of the metabolites are then analyzed within clusters. We created two freely available applications to assist the workflow: FragClust to generate the tables to be subjected to CHCA, and TestClust to perform statistical analysis on clustered data. We used the lipidomics data from the plasma of Alzheimer's disease (AD) patients in comparison with healthy controls to test the workflow. To date, the search for plasma biomarkers in AD has not provided reliable results. This article shows that the workflow is helpful to understand the behavior of whole lipid classes in plasma of AD patients.

Albumin versus solvent/detergent-treated pooled plasma as replacement fluid for long-term plasma exchange therapy in a patient with primary hypertriglyceridemia and recurrent hyperlipidemic pancreatitis.

BACKGROUND: Chylomicronemia syndrome is a metabolic condition characterized by severe fasting hypertrigliceridemia (≥ 1000 mg/dL) and other clinical features including chronic abdominal pain and recurrent acute pancreatitis. In patients with acute or recurrent pancreatitis, plasma exchange (PEx) is indicated for the treatment of acute disease and prevention of recurrence. The use of plasma instead of albumin as replacement fluid has been suggested for its putative ability to replace the deficient enzyme possibly leading to better clinical improvement. CASE REPORT: A 40-year-old man with chylomicronemia syndrome due to a newly identified loss-of-function mutation in the lipoprotein lipase (LPL) gene (IVS2, c.250-1G/C) has been treated at our hospital since the age of 13. From age 18 to age 34, the patient had five episodes of acute pancreatitis while his triglyceride (TG) levels were extremely high (2500-4000 mg/dL). As the TG levels remained stable over 4000 mg/dL despite the maximum medical treatment, the patient started long-term PEx treatment on a weekly basIs. Both albumin and plasma have been used as replacement solution. Thirty months from the beginning of this treatment, no episode of acute pancreatitis has been reported, and the chronic abdominal pain fully disappeared. No differences were observed between the use of albumin or plasma as replacement solution. CONCLUSION: Long-term PEx is effective in preventing the recurrence of acute pancreatitis and in treatment of chronic abdominal pain in this patient with chylomicronemia syndrome. Plasma is not more effective than albumin in lipid reduction, likely because of its extremely low enzyme content. Therefore, in patients with LPL deficiency serum albumin should be preferred to plasma as replacement fluid because of the low rate of side effects and reduced costs.