Lipid traits and lipid-lowering drug target genes and risk of melanoma: a mendelian randomization study.

Our study aimed to investigate the role of lipids in melanoma risk and the effect of lipid-lowering drug targets on melanoma. Using Mendelian Randomization analysis, we examined the genetic agents of nine lipid-lowering drugs and their association with melanoma risk. We found that genetically proxied inhibition of HMGCR, ABCG5/ABCG8, and ANGPTL3 was associated with a reduced risk of melanoma. On the other hand, inhibition of LPL and Apo-B100 was significantly associated with an increased risk of melanoma. Sensitivity analyses did not reveal any statistical evidence of bias from pleiotropy or genetic confounding. We did not find a robust association between lipid traits NPC1L1, PCSK9, APOC3 inhibition, and melanoma risk. These findings were validated using two independent lipid datasets. Our analysis also revealed that HMGCR, ANGPTL3, and ABCG5/ABCG8 inhibitors reduced melanoma risk independent of their effects on lipids. This suggests that these targets may have potential for melanoma prevention or treatment. In conclusion, our study provides evidence for a causal role of lipids in melanoma risk and highlights specific lipid-lowering drug targets that may be effective in reducing the risk of melanoma. These findings contribute to the understanding of the underlying mechanisms of melanoma development and provide potential avenues for further research and therapeutic interventions.

Heat-killed Lactiplantibacillus plantarum Shinshu N-07 exerts antiobesity effects in western diet-induced obese mice.

AIMS: The aim of this study was to evaluate the antiobesity effects of heat-killed Lactiplantibacillus plantarum Shinshu N-07 (N-07) isolated from fermented Brassica rapa L. METHODS AND RESULTS: Male mice were divided into three groups (n = 10/group); normal diet, western diet (WD), or WD + N-07 (N-07) group and administered each diet for 56 days. The N-07 group showed significant suppression of body weight gain and epididymal fat, perirenal fat, and liver weights compared with the WD group. Higher levels of fecal total cholesterol, triglyceride (TG), and free fatty acid (FFA) were observed in the N-07 group than in the WD group. The mRNA expression of the cholesterol transporter ATP-binding cassette transporter G5 (ABCG5) was significantly increased in the small intestine of N-07-fed mice compared with WD-fed mice. Moreover, N-07 supplementation significantly increased the mRNA expression of ABCG5 and ABCG8 in Caco-2 cells. Furthermore, the TG- and FFA-removal ability of N-07 was confirmed to evaluate its soybean oil- and oleic acid-binding capacities in in vitro experiments. CONCLUSIONS: The antiobesity effects of N-07 might be due to its ability to promote lipid excretion by regulating cholesterol transporter expression and lipid-binding ability.

Associations of Dietary Cholesterol Consumption With Incident Diabetes and Cardiovascular Disease: The Role of Genetic Variability in Cholesterol Absorption and Disease Predisposition.

OBJECTIVE: Whether genetic susceptibility to disease and dietary cholesterol (DC) absorption contribute to inconsistent associations of DC consumption with diabetes and cardiovascular disease (CVD) remains unclear. RESEARCH DESIGN AND METHODS: DC consumption was assessed by repeated 24-h dietary recalls in the UK Biobank. A polygenetic risk score (PRS) for DC absorption was constructed using genetic variants in the Niemann-Pick C1-Like 1 and ATP Binding Cassettes G5 and G8 genes. PRSs for diabetes, coronary artery disease, and stroke were also created. The associations of DC consumption with incident diabetes (n = 96,826) and CVD (n = 94,536) in the overall sample and by PRS subgroups were evaluated using adjusted Cox models. RESULTS: Each additional 300 mg/day of DC consumption was associated with incident diabetes (hazard ratio [HR], 1.17 [95% CI, 1.07-1.27]) and CVD (HR, 1.09 [95% CI, 1.03-1.17]), but further adjusting for BMI nullified these associations (HR for diabetes, 0.99 [95% CI, 0.90-1.09]; HR for CVD, 1.04 [95% CI, 0.98-1.12]). Genetic susceptibility to the diseases did not modify these associations (P for interaction ≥0.06). The DC-CVD association appeared to be stronger in people with greater genetic susceptibility to cholesterol absorption assessed by the non-high-density lipoprotein cholesterol-related PRS (P for interaction = 0.04), but the stratum-level association estimates were not statistically significant. CONCLUSIONS: DC consumption was not associated with incident diabetes and CVD, after adjusting for BMI, in the overall sample and in subgroups stratified by genetic predisposition to cholesterol absorption and the diseases. Nevertheless, whether genetic predisposition to cholesterol absorption modifies the DC-CVD association requires further investigation.

Gene variants and clinical characteristics of children with sitosterolemia.

OBJECTIVE: To enhance the detection, management and monitoring of Chinese children afflicted with sitosterolemia by examining the physical characteristics and genetic makeup of pediatric patients. METHODS: In this group, 26 children were diagnosed with sitosterolemia, 24 of whom underwent genetic analysis. Patient family medical history, physical symptoms, tests for liver function, lipid levels, standard blood tests, phytosterol levels, cardiac/carotid artery ultrasounds, fundus examinations, and treatment were collected. RESULTS: The majority (19, 73.1%) of the 26 patients exhibited xanthomas as the most prevalent manifestation. The second most common symptoms were joint pain (7, 26.9%) and stunted growth (4, 15.4%). Among the 24 (92.3%) patients whose genetics were analyzed, 16 (66.7%) harbored ABCG5 variants (type 2 sitosterolemia), and nearly one-third (8, 33.3%) harbored ABCG8 variants (type 1 sitosterolemia). Additionally, the most common pathogenic ABCG5 variant was c.1166G > A (p.Arg389His), which was found in 10 patients (66.7%). Further analysis did not indicate any significant differences in pathological traits among those carrying ABCG5 and ABCG8 variations (P > 0.05). Interestingly, there was a greater abundance of nonsense variations in ABCG5 than in ABCG8 (P = 0.09), and a greater frequency of splicing variations in ABCG8 than ABCG5 (P = 0.01). Following a change in diet or a combination of ezetimibe, the levels of cholesterol and low-density lipoprotein were markedly decreased compared to the levels reported before treatment. CONCLUSION: Sitosterolemia should be considered for individuals presenting with xanthomas and increased cholesterol levels. Phytosterol testing and genetic analysis are important for early detection. Managing one's diet and taking ezetimibe can well control blood lipids.

Platelet proteomic profiling in sitosterolemia suggests thrombocytopenia is driven by lipid disorder and not platelet aberrations.

ABSTRACT: Sitosterolemia is a rare autosomal recessive genetic disorder in which patients develop hypercholesterolemia and may exhibit abnormal hematologic and/or liver test results. In this disease, dysfunction of either ABCG5 or ABCG8 results in the intestinal hyperabsorption of all sterols, including cholesterol and, more specifically, plant sterols or xenosterols, as well as in the impaired ability to excrete xenosterols into the bile. It remains unknown how and why some patients develop hematologic abnormalities. Only a few unrelated patients with hematologic abnormalities at the time of diagnosis have been reported. Here, we report on 2 unrelated pedigrees who were believed to have chronic immune thrombocytopenia as their most prominent feature. Both consanguineous families showed recessive gene variants in ABCG5, which were associated with the disease by in silico protein structure analysis and clinical segregation. Hepatosplenomegaly was absent. Thrombopoietin levels and megakaryocyte numbers in the bone marrow were normal. Metabolic analysis confirmed the presence of strongly elevated plasma levels of xenosterols. Potential platelet proteomic aberrations were longitudinally assessed following dietary restrictions combined with administration of the sterol absorption inhibitor ezetimibe. No significant effects on platelet protein content before and after the onset of treatment were demonstrated. Although we cannot exclude that lipotoxicity has a direct and platelet-specific impact in patients with sitosterolemia, our data suggest that thrombocytopenia is neither caused by a lack of megakaryocytes nor driven by proteomic aberrations in the platelets themselves.

Weighted burden analysis of rare coding variants in 470,000 exome-sequenced UK Biobank participants characterises effects on hyperlipidaemia risk.

A previous study of 200,000 exome-sequenced UK Biobank participants investigating the association between rare coding variants and hyperlipidaemia had implicated four genes, LDLR, PCSK9, APOC3 and IFITM5, at exome-wide significance. In addition, a further 43 protein-coding genes were significant with an uncorrected p value of <0.001. Exome sequence data has become available for a further 270,000 participants and weighted burden analysis to test for association with hyperlipidaemia was carried out in this sample for the 47 genes highlighted by the previous study. There was no evidence to implicate IFITM5 but LDLR, PCSK9, APOC3, ANGPTL3, ABCG5 and NPC1L1 were all statistically significant after correction for multiple testing. These six genes were also all exome-wide significant in the combined sample of 470,000 participants. Variants impairing function of LDLR and ABCG5 were associated with increased risk whereas variants in the other genes were protective. Variant categories associated with large effect sizes are cumulatively very rare and the main benefit of this kind of study seems to be to throw light on the molecular mechanisms impacting hyperlipidaemia risk, hopefully supporting attempts to develop improved therapies.

Plasma campesterol and ABCG5/ABCG8 gene loci on the risk of cholelithiasis and cholecystitis: evidence from Mendelian randomization and colocalization analyses.

The causal relationships between plasma metabolites and cholelithiasis/cholecystitis risks remain elusive. Using two-sample Mendelian randomization, we found that genetic proxied plasma campesterol level showed negative correlation with the risk of both cholelithiasis and cholecystitis. Furthermore, the increased risk of cholelithiasis is correlating with the increased level of plasma campesterol. Lastly, genetic colocalization study showed that the leading SNP, rs4299376, which residing at the ABCG5/ABCG8 gene loci, was shared by plasma campesterol level and cholelithiasis, indicating that the aberrant transportation of plant sterol/cholesterol from the blood stream to the bile duct/gut lumen might be the key in preventing cholesterol gallstone formation.

A Clinical Case of Probable Sitosterolemia.

Sitosterolemia is a rare genetic lipid disorder characterized by elevated plant sterols in the serum. A 24-year-old Japanese woman was referred to our hospital due to a high serum low-density lipoprotein cholesterol (LDL-C) level of 332 mg/dL. At first, she was suspected to suffer from familial hypercholesterolemia, and thus received lipid-lowering agents. Although her LDL-C level remained high (220 mg/dL) with diet therapy plus 10 mg/day rosuvastatin, it was drastically decreased to 46 mg/dL with the addition of 10 mg/day ezetimibe. Finally, her LDL-C level was well-controlled at about 70 mg/dL with 10 mg/day ezetimibe alone. Furthermore, while her serum sitosterol level was elevated at 10.5 µg/mL during the first visit to our hospital, it decreased to 3.6 µg/mL with the 10 mg/day ezetimibe treatment alone. These observations suggest that she might probably suffer from sitosterolemia. Therefore, targeted gene sequencing analysis was performed using custom panels focusing on the exome regions of 21 lipid-associated genes, including ABCG5, ABCG8, and familial hypercholesterolemia-causing genes (LDL receptor, LDLRAP1, PCSK9, and apolipoprotein B). We finally identified a heterozygous ABCG8 variant (NM_022437.2:c.1285A>G or NP_071882.1:p.Met429Val) in our patient. The same gene mutation was detected in her mother. We report here a rare case exhibiting probable sitosterolemia caused by a heterozygous Met429Val variant in the ABCG8 gene and additional unknown variants.

LRH-1 agonist DLPC through STAT6 promotes macrophage polarization and prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition-associated cholestasis (PNAC) is an important complication in patients with intestinal failure with reduced LRH-1 expression. Here, we hypothesized that LRH-1 activation by its agonist, dilauroylphosphatidylcholine (DLPC), would trigger signal transducer and activator of transcription 6 (STAT6) signaling and hepatic macrophage polarization that would mediate hepatic protection in PNAC. APPROACH AND RESULTS: PNAC mouse model (oral DSSx4d followed by PNx14d; DSS-PN) was treated with LRH-1 agonist DLPC (30 mg/kg/day) intravenously. DLPC treatment prevented liver injury and cholestasis while inducing hepatic mRNA expression of Nr5a2 (nuclear receptor subfamily 5 group A member 2), Abcb11 (ATP binding cassette subfamily B member 11), Abcg5 (ATP-binding cassette [ABC] transporters subfamily G member 5), Abcg8 (ATP-binding cassette [ABC] transporters subfamily G member 8), nuclear receptor subfamily 0, and ATP-binding cassette subfamily C member 2 ( Abcc2) mRNA, all of which were reduced in PNAC mice. To determine the mechanism of the DLPC effect, we performed RNA-sequencing analysis of the liver from Chow, DSS-PN, and DSS-PN/DLPC mice, which revealed DLPC upregulation of the anti-inflammatory STAT6 pathway. In intrahepatic mononuclear cells or bone-marrow derived macrophages (BMDM) from PNAC mice, DLPC treatment prevented upregulation of pro-inflammatory (M1) genes, suppressed activation of NFκB and induced phosphorylation of STAT6 and its target genes, indicating M2 macrophage polarization. In vitro, incubation of DLPC with cultured macrophages showed that the increased Il-1b and Tnf induced by exposure to lipopolysaccharides or phytosterols was reduced significantly, which was associated with increased STAT6 binding to promoters of its target genes. Suppression of STAT6 expression by siRNA in THP-1 cells exposed to lipopolysaccharides, phytosterols, or both resulted in enhanced elevation of IL-1B mRNA expression. Furthermore, the protective effect of DLPC in THP-1 cells was abrogated by STAT6 siRNA. CONCLUSIONS: These results indicate that activation of LRH-1 by DLPC may protect from PNAC liver injury through STAT6-mediated macrophage polarization.

Molecular and computational characterization of ABCB11 and ABCG5 variants in Tunisian patients with neonatal/infantile low-GGT intrahepatic cholestasis: Genetic diagnosis and genotype-phenotype correlation assessment.

Many inherited conditions cause hepatocellular cholestasis in infancy, including progressive familial intrahepatic cholestasis (PFIC), a heterogeneous group of diseases with highly overlapping symptoms. In our study, six unrelated Tunisian infants with PFIC suspicion were the subject of a panel-target sequencing followed by an exhaustive bioinformatic and modeling investigations. Results revealed five disease-causative variants including known ones: (the p.Asp482Gly and p.Tyr354 * in the ABCB11 gene and the p.Arg446 * in the ABCC2 gene), a novel p.Ala98Cys variant in the ATP-binding cassette subfamily G member 5 (ABCG5) gene and a first homozygous description of the p.Gln312His in the ABCB11 gene. The p.Gln312His disrupts the interaction pattern of the bile salt export pump as well as the flexibility of the second intracellular loop domain harboring this residue. As for the p.Ala98Cys, it modulates both the interactions within the first nucleotide-binding domain of the bile transporter and its accessibility. Two additional potentially modifier variants in cholestasis-associated genes were retained based on their pathogenicity (p.Gly758Val in the ABCC2 gene) and functionality (p.Asp19His in the ABCG8 gene). Molecular findings allowed a PFIC2 diagnosis in five patients and an unexpected diagnosis of sisterolemia in one case. The absence of genotype/phenotype correlation suggests the implication of environmental and epigenetic factors as well as modifier variants involved directly or indirectly in the bile composition, which could explain the cholestasis phenotypic variability.

Air channels create a directional light signal to regulate hypocotyl phototropism.

In plants, light direction is perceived by the phototropin photoreceptors, which trigger directional growth responses known as phototropism. The formation of a phototropin activation gradient across a photosensitive organ initiates this response. However, the optical tissue properties that functionally contribute to phototropism remain unclear. In this work, we show that intercellular air channels limit light transmittance through various organs in several species. Air channels enhance light scattering in Arabidopsis hypocotyls, thereby steepening the light gradient. This is required for an efficient phototropic response in Arabidopsis and Brassica. We identified an embryonically expressed ABC transporter required for the presence of air channels in seedlings and a structure surrounding them. Our work provides insights into intercellular air space development or maintenance and identifies a mechanism of directional light sensing in plants.

Genetic analysis and functional study of a novel ABCG5 mutation in sitosterolemia with hematologic disease.

Sitosterolemia is a rare autosomal recessive hereditary disease caused by loss-of-function genetic mutations in either ATP-binding cassette subfamily G member 5 or member 8 (ABCG5 or ABCG8). Here, we investigate novel variants in ABCG5 and ABCG8 that are associated with the sitosterolemia phenotype. We describe a 32-year-old woman with hypercholesterolemia, tendon and hip xanthomas, autoimmune hemolytic anemia and macrothrombocytopenia from early life, which make us highly suspicious of the possibility of sitosterolemia. A novel homozygous variant in ABCG5 (c.1769C>A, p.S590X) was identified by genomic sequencing. We also examined the lipid profile, especially plant sterols levels, using gas chromatography-mass spectrometry. Functional studies, including western blotting and immunofluorescence staining, showed that the nonsense mutation ABCG5 1769C>A hinders the formation of ABCG5 and ABCG8 heterodimers and the function of transporting sterols. Our study expands the knowledge of variants in sitosterolemia and provides diagnosis and treatment recommendations.

Macrophage inhibitory cytokine-1 aggravates diet-induced gallstone formation via increased ABCG5/ABCG8 expression.

Macrophage inhibitory cytokine 1 (MIC-1), which is overproduced in various human cancers and associated with cachexia, acts on the hypothalamus to suppress appetite and reduce body weight. We investigated the mechanisms through which MIC-1 affects bile acid metabolism and gallstone formation, which are poorly understood. Over 6 weeks, male C57BL/6 mice fed either standard chow or a lithogenic diet were intraperitoneally injected with phosphate-buffered saline (PBS) or MIC-1 (200 µg/kg/week). Among lithogenic diet-fed mice, MIC-1 treatment resulted in increased gallstone formation compared with PBS treatment. Compared with PBS treatment, MIC-1 treatment decreased hepatic cholesterol and bile acid levels and reduced expression of HMG-CoA reductase (HMGCR), the master cholesterol metabolism regulator sterol regulatory element-binding protein 2, cholesterol 7α-hydroxylase (CYP7A1), mitochondrial sterol 27-hydroxylase, and oxysterol 7α-hydroxylase. Compared with PBS treatment, MIC-1 treatment had no effect on small heterodimer partner, farnesoid X receptor, or pregnane X receptor expression, and extracellular signal-related kinase and c-Jun N-terminal kinase phosphorylation decreased, suggesting that these factors do not contribute to the MIC-1-induced reduction in CYP7A1 expression. Compared with PBS treatment, MIC-1 treatment increased AMP-activated protein kinase (AMPK) phosphorylation. Treatment with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reduced CYP7A1 and HMGCR expression, whereas the AMPK inhibitor Compound C reversed MIC-1-induced reductions in CYP7A1 and HMGCR expression. Furthermore, in MIC-1-treated mice, total biliary cholesterol levels increased together with increased ATP-binding cassette subfamily G (ABCG)5 and ABCG8 expression. Compared with PBS treatment, MIC-1 treatment did not affect expression of liver X receptors α and ß, liver receptor homolog 1, hepatocyte nuclear factor 4α, or NR1I3 (also known as constitutive androstane receptor), which are upstream of ABCG5/8; however, MIC-1 treatment increased ABCG5/8 expression and promoter activities. Our study indicates that MIC-1 influences gallstone formation by increasing AMPK phosphorylation, reducing CYP7A1 and HMGCR expression, and increasing ABCG5 and ABCG8 expression.

Differential Effects of ABCG5/G8 Gene Region Variants on Lipid Profile, Blood Pressure Status, and Gallstone Disease History in Taiwan.

ABCG5 and ABCG8 are two key adenosine triphosphate-binding cassette (ABC) proteins that regulate whole-body sterol trafficking. This study aimed to elucidate the association between ABCG5/G8 gene region variants and lipid profile, cardiometabolic traits, and gallstone disease history in Taiwan. A total of 1494 Taiwan Biobank participants with whole-genome sequencing data and 117,679 participants with Axiom Genome-Wide CHB Array data were enrolled for analysis. Using genotype-phenotype and stepwise linear regression analyses, we found independent associations of four Asian-specific ABCG5 variants, rs119480069, rs199984328, rs560839317, and rs748096191, with total, low-density lipoprotein (LDL), and non-high-density lipoprotein (HDL) cholesterol levels (all p ≤ 0.0002). Four other variants, which were in nearly complete linkage disequilibrium, exhibited genome-wide significant associations with gallstone disease history, and the ABCG8 rs11887534 variant showed a trend of superiority for gallstone disease history in a nested logistic regression model (p = 0.074). Through regional association analysis of various other cardiometabolic traits, two variants of the PLEKHH2, approximately 50 kb from the ABCG5/G8 region, exhibited significant associations with blood pressure status (p < 10-6). In conclusion, differential effects of ABCG5/G8 region variants were noted for lipid profile, blood pressure status, and gallstone disease history in Taiwan. These results indicate the crucial role of individualized assessment of ABCG5/G8 variants for different cardiometabolic phenotypes.

Children with Severe Hypercholesterolemia Caused by a Pathogenic Mutation in ABCG5.

We herein present a case series of hypercholesterolemia caused by a pathogenic mutation in the ATP-binding cassette sub-family G member 5 (ABCG5). Three unrelated infantile patients who were breastfed and had extremely elevated low-density lipoprotein (LDL) cholesterol levels were referred to our hospital. Their LDL cholesterol levels decreased significantly after weaning. Panel sequencing revealed a pathogenic mutation in ABCG5 in each patient. An 8-year-old girl was also referred due to suspected familial hypercholesterolemia. Panel sequencing revealed a pathogenic mutation in ABCG5. A cholesterol-reduced diet alone significantly reduced the LDL cholesterol levels. Moreover, the administration of ezetimibe was found to be beneficial.

Sitosterolemia.

Sitosterolemia is an inherited metabolic disorder characterized by increased levels of plant sterols, such as sitosterol. This disease is caused by loss-of-function genetic mutations in the ATP-binding cassette (ABC) subfamily G member 5 or member 8 (ABCG5 or ABCG8, respectively), both of which play important roles in the selective excretion of plant sterols from the liver and intestine, leading to a failure to excrete plant sterols. Sitosterolemia, which is currently considered a rare genetic disorder, has been described as a phenocopy of homozygous familial hypercholesterolemia (FH). Typical phenotypes of sitosterolemia, including elevated low-density lipoprotein (LDL) cholesterol, tendon xanthomas, and premature coronary artery disease, overlap those of homozygous FH; however, there are substantial differences between these two diseases in terms of treatments and prognoses. Moreover, it is of note that sitosterolemia appears to be quite underdiagnosed, although accurate diagnosis and appropriate interventions will likely to lead to better prognoses compared with homozygous FH. Unlike cases of homozygous FH, dietary counseling is quite effective in reducing the LDL cholesterol as well as sitosterol of patients with sitosterolemia. In this chapter, we summarize the current understandings of this disease and provide useful tips for the diagnosis as well as better treatment of patients with sitosterolemia.

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8.

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.

Plant sterol hyperabsorption caused by uncontrolled diabetes in a patient with a heterozygous ABCG5 variant.

Plant sterol intake is widely recommended for patients with cardiovascular risk factors based on the inhibitory effect on intestinal cholesterol absorption. Although plant sterols, once absorbed, can promote atherosclerosis, their intake is believed to be safe because of poor absorption, except in rare hyperabsorbers with homozygous ABCG5/8 mutations. We report a case of new-onset type 1 diabetes accompanied by hypercholesterolemia. At the initial presentation with diabetic ketoacidosis, the patient showed marked hypercholesterolemia. Whole-exome sequencing revealed a heterozygous pathogenic variant in ABCG5 (p.R419H). The initial serum plant sterol levels were markedly high (sitosterol 32.5 µg/mL, campesterol 66.0 µg/mL), close to the range observed in patients with homozygous ABCG5/8 mutations, which were largely reduced by insulin treatment without ezetimibe. The addition of ezetimibe normalized plant sterol levels. These findings provide the first evidence that uncontrolled diabetes plays a causal role in the pathogenesis of phytosterolemia.

Screening of ABCG5 and ABCG8 Genes for Sitosterolemia in a Familial Hypercholesterolemia Cascade Screening Program.

BACKGROUND: Sitosterolemia is a rare autosomal recessive disorder caused by homozygous or compound heterozygous variants in ABCG5/ABCG8. The disease is characterized by increased plasma plant sterols. Small case series suggest that patients with sitosterolemia have wide phenotypic heterogeneity with great variability on either plasma cholesterol levels or development of atherosclerotic cardiovascular disease. The present study aims to characterize the prevalence and clinical features of sitosterolemia participating in a familial hypercholesterolemia genetic cascade screening program. METHODS: From 443 familial hypercholesterolemia index cases, 260 were negative for familial hypercholesterolemia genes and were sequenced for the ABCG5/8 genes. Clinical and laboratory characteristics of affected individuals were determined. RESULTS: Eight (3.1%) index cases were found to be homozygous or compound heterozygous variant for ABCG5/ABCG8 genes, confirming the genetic diagnosis of sitosterolemia. Screening their relatives led to the identification of 6 additional confirmed sitosterolemia cases (3 homozygous and 3 compound heterozygous variant) and 18 carriers (heterozygous). The mean age of identified sitosterolemia cases (n=14) was 37.2±19.8 years, 50% were females, and 78.6% (all adults) presented either clinical or subclinical atherosclerotic cardiovascular disease. As expected, affected individuals presented elevated plasma plant sterol levels (mean ß-Sitosterol and campesterol, respectively, 160.3±107.1 and 32.0±19.6 µg/mL) and the highest plasma LDL (low-density lipoprotein)-cholesterol was 269.0±120.0 mg/dL (range: 122-521 mg/dL). LDL-cholesterol mean reduction with therapy among cases was 65%. Eighty-three percent (83%) of identified sitosterolemia patients presented hematologic abnormalities. CONCLUSIONS: Testing genes associated with sitosterolemia in the molecular routine workflow of a familial hypercholesterolemia cascade screening program allowed the precise diagnosis of sitosterolemia in a substantial number of patients with varying LDL-C levels and high incidence of early atherosclerotic cardiovascular disease and hematologic abnormalities.

Evolutionary origin and sequence signatures of the heterodimeric ABCG5/ABCG8 transporter.

ATP-binding cassette (ABC) systems, characterized by ABC-type nucleotide-binding domains (NBDs), play crucial roles in various aspects of human physiology. Human ABCG5 and ABCG8 form a heterodimeric transporter that functions in the efflux of sterols. We used sequence similarity search, multiple sequence alignment, phylogenetic analysis, and structure comparison to study the evolutionary origin and sequence signatures of ABCG5 and ABCG8. Orthologs of ABCG5 and ABCG8, supported by phylogenetic analysis and signature residues, were identified in bilaterian animals, Filasterea, Fungi, and Amoebozoa. Such a phylogenetic distribution suggests that ABCG5 and ABCG8 could have originated in the last common ancestor of Amorphea (the unikonts), the eukaryotic group including Amoebozoa and Opisthokonta. ABCG5 and ABCG8 were missing in genomes of various lineages such as snakes, jawless vertebrates, non-vertebrate chordates, echinoderms, and basal metazoan groups. Amino-acid changes in key positions in ABCG8 Walker A motif and/or ABCG5 C-loop were observed in most tetrapod organisms, likely resulted in the loss of ATPase activity at one nucleotide-binding site. ABCG5 and ABCG8 in Ecdysozoa (such as insects) exhibit elevated evolutionary rates and accumulate various changes in their NBD functional motifs. Alignment inspection revealed several residue positions that show different amino-acid usages in ABCG5/ABCG8 compared to other ABCG subfamily proteins. These residues were mapped to the structural cores of transmembrane domains (TMDs), the NBD-TMD interface, and the interface between TMDs. They serve as sequence signatures to differentiate ABCG5/ABCG8 from other ABCG subfamily proteins, and some of them may contribute to substrate specificity of the ABCG5/ABCG8 transporter.