ABCG5 Mutations Research Articles

A Descriptive Analysis of Patients with ABCG8 Mutation and Anemia. a Meta-Analysis and Systematic Review of an Uncommon Cause of Unexplained Anemia

Background Sitosterolemia is a rare inherited condition caused by elevated levels of plant sterols in the plasma. The genetic basis of the condition is characterized by homozygous and compound heterozygous mutations in ABCG5 and ABCG8 genes. The condition is very rare that information about this in literature is limited to case reports, case series and translational observational studies. A scarce occurrence in this already infrequent condition are hematological abnormalities such as hemolytic anemia, stomatocytosis, and macrothrombocytopenia. Basic details of the disease such are frequency of occurrence, demographic details of patients, and degree and type of anemia are not available. We conducted a meta-analysis and systematic review to answer these questions regarding patients who have hemolytic anemia and ABCG8 mutation. The study was also prompted by a clinical experience that our group encountered, of an adult male who had chronic severe hemolysis, at times requiring transfusion support, in whom, extensive investigation failed to reveal a cause for the hemolysis. The patient was ultimately found to have a mutation in the ABCG8 gene. Methods A controlled vocabulary encompassing ABCG8, sitosterolemia, phytosterolemia, anemia and hemolysis was used to perform a systematic search in PubMed, Embase, Scopus and Cochrane, from inception to July 2023. No restrictions on date, language, status, or outcomes were applied. 532 records were identified after duplicates were removed using Mendeley 1.19.8. The titles and abstracts were screened independently by 2 reviewers. We only included studies pertaining to patients with sitosterolemia, ABCG8 mutation and anemia of any cause or severity. Studies associated with ABCG5 mutation alone or lacked anemia were excluded. 13 reports were shortlisted for the final analysis (Observational studies-6, case series-4, case reports-3). Descriptive statistics were utilized to study the patient characteristics. The proportion of patients having both anemia and any ABCG8 mutation among all cases of sitosterolemia was determined using the DerSimonian-Laird random effects model with OpenMeta. Results From the 13 reports that we found in available literature, we identified 19 cases of ABCG8 mutation and anemia. From the random-effects proportions model, the chance of this event occurring among patients with sitosterolemia was 6.8% [0.068, 95% Confidence Interval (CI) 0.016-0.120, p=0.010] (I 2 24.68%) (14/145) (figure 1). Various characteristics of the cohort analyzed, including the different ABCG8 mutations, are summarized in figure 2. The mean age of the patients was 26 years (Age range: 0.1-61 years) and included neonates, pediatric, and adult patients. There were 7 females and 7 males among those whose sex were reported. 9/13 studies documented elevated sterols, while 8/13 clearly stated hemolysis as the cause of anemia. The hemoglobin ranged between 6.1 to 12.6 g/dl with a mean of 10.2 g/dl. Thrombocytopenia and stomatocytosis were frequently reported. Splenomegaly and xanthomas were other common associations. One example demonstrated an improvement in anemia after ezetimibe therapy, while splenectomy and supportive care was utilized in a couple of other reports to treat hemolysis. Conclusions To the best of our knowledge, we provide the first report of the prevalence of anemia, specifically in patients with sitosterolemia caused by a mutation in the ABCG8 gene. At 6.8%, this is an extremely rare occurrence in an already infrequent disease. A high index of suspicion is required to arrive at this diagnosis. The age distribution demonstrates a peculiar aspect of the disease, that it can occur very early in infancy, as well as in adults, likely triggered by environmental factors in an already predisposed patient. Unexplained hemolytic anemia, especially if associated with thrombocytopenia, splenomegaly and xanthomas should prompt testing for sterol levels and genomic sequencing to identify pathogenic variants of ABCG8 or ABCG5. The degree of anemia was mild in our analysis and hence unlikely to lead to significant mortality or morbidity. Heterogeneity between the various reports is a limitation of our analysis. While we specifically focused on ABCG8 and anemia, future research can also include ABCG5 related anemia and attempt to compare the two cohorts.

Abstract 447: Cholesterol Excretion In Whole-body And Liver-specific ABCG5 ABCG8-deficient Mice

Background: The ABCG5 ABCG8 sterol transporter is the body’s primary defense against the accumulation of dietary sterols. Loss of function mutations in ABCG5 or ABCG8 result in Sitosterolemia, a rare disorder that presents as familial hypercholesterolemia, but is distinguished by recessive genetics and the accumulation of phytosterols in plasma and tissues. Biliary cholesterol is reduced by greater than 80% in ABCG5 ABCG8-deficient mice, but fecal neutral sterols are unaffected, suggesting a G5G8-independent pathway for cholesterol excretion. The objectives of the present study were to determine the ability of whole-body and liver-specific G5G8-deficient mice to maintain fecal neutral sterol output when challenged with cholesterol -enriched diet and determine maximal rates of biliary cholesterol secretion. Methods: Wild-type, whole-body and liver-specific ABCG5 ABCG8-deficient mice were maintained on cereal-based mixed diet, a phytosterol-free purified diet, or a purified diet supplemented with 0.2% cholesterol. Following acclimatization to each diet, fecal neutral sterol output was determined over a three-day period by gas chromatography/mass spectrometry. Maximal biliary cholesterol secretion rates were determined by biliary diversion with bile acid infusion over a range of 0 to 1000 nmol/minute. Biliary lipid secretion was determined by enzymatic colorimetric assays and molecular coupling of cholesterol to bile acid and phospholipid secretion calculated by linear regression. Results & Conclusions: Whole-body G5G8-deficient mice exhibited a 40% reduction in fecal neutral sterol output compared to wild-type mice when maintained on cereal-based or purified, low cholesterol containing diets. Cholesterol feeding resulted in a 10-fold increase in fecal neutral sterols in wild-type mice, an effect not observed in G5G8-deficient mice. These data indicate that ABCG5 ABCG8 is indispensable in opposing the accumulation of dietary cholesterol and that alternative pathways cannot compensate for G5G8 deficiency.

*An Unsolved Case: FH or Sitosterolemia?

<h3>Lead Author's Financial Disclosures</h3> Nothing to disclose. <h3>Study Funding</h3> None. <h3>Background/Synopsis</h3> A 51-year-old woman with diabetes mellitus (A1c 6.7%), obesity (BMI 34), hyperlipidemia, and recurrent choledocholithiasis pending cholecystectomy, presents to UVA Prevention Clinic. Hyperlipidemia was diagnosed at age 22. Cholesterols have ranged from 290 to 520ng/dL on rosuvastatin 40mg daily. Family history was negative for premature coronary artery disease, but notable for high cholesterol in her mother and teenage child. Exam demonstrated left eyelid xanthelasma and red-yellow scaly patch on her left elbow. Labs revealed cholesterol 400ng/dL, LDL 307ng/dL, HDL 43ng/dL, triglycerides 223ng/dL, and HgA1c 6.7%. Colestipol 1mg BID and ezetimibe 10mg daily were initiated. Follow-up labs demonstrated cholesterol 284ng/dL, HDL 43ng/dL, LDL 199ng/dL and triglycerides 199ng/dL. Genetic testing for familial hypercholesteremia (FH) revealed an Increased Risk Allele on ABCG8 (c.55G>C, p.Asp19His) and a Variant of Uncertain Significance on ABCG8 f(c.275G>A, p.Ser92Asn). Subsequent sitosterol level testing, on medications, was 4.3mg/L (normal < 6.0). <h3>Objective/Purpose</h3> This abstract serves as an educational unsolved clinical vignette. <h3>Methods</h3> Sitosterolemia is an autosomal recessive disorder of lipid metabolism characterized by net increased intestinal absorption via decreased biliary excretion of plant sterols. Sitosterolemia has considerable overlap with FH but is significantly rarer1. Both cause xanthomas and premature coronary atherosclerosis sitosterolemia presentation also causes choledocholithiasis, hemolysis, splenomegaly, and arthralgia/arthritis. LDL levels in FH are consistently high whereas in sitosterolemia levels vary considerably with diet. <h3>Results</h3> Diagnostic criteria for sitosterolemia is comprised of four categories: clinical manifestations, serum sitosterol levels, exclusion of FH and cerebroteinous xanthomatosis, and pathogenic mutations. For definitive diagnosis, pathogenic mutations in ABCG5 or ABCG8 must be identified. Accurate diagnosis is changing management. Lower intake of plant sterol rich foods are recommended in sitosterolemia, foods usually considered beneficial in FH3. Ezetimibe and bile-acid sequestrants are shown to reduce sitosterol and cholesterol levels through decreased intestinal sterol absorption. <h3>Conclusions</h3> The patient described above has findings possibly consistent with sitosterolemia. Her Dutch FH score of 9, used for clinical diagnosis of FH, is consistent with definitive FH. The diagnostic dilemma has multiple potential approaches: A) Stopping colestipol and ezetimibe, liberalizing diet, and retesting sitosterol levels. B) Continuing current medications, reinforcing a traditional heart-healthy diet, and adding a PCSK-9i for presumed FH. C) Continuing current medications, adding a PCSK-9i for presumed FH, but encouraging a sitosterolemia diet low in plant sterols. D) Continuing current medications, reinforcing a sitosterolemia diet low in plant sterols, and rechecking lipids in 3-4 months.

*A Novel ABCG8 Variant and Apo(A) Short Isoform Associated with Premature Atherosclerosis and Familial Hypercholesterolemia Phenocopy.

Lead Author's Financial Disclosures Nothing to disclose. Study Funding None. Background/Synopsis We report a patient with a rare ABCG8 mutation, that when combined with Apo(a) short isoforms may represent a familial hypercholesterolemia (FH) phenocopy. Objective/Purpose A 44-year-old African American male with prior myocardial infarction (MI) at the age 24 with subsequent coronary artery bypass graft who had rapid progression of atherosclerosis despite having a corrected LDL of zero. Methods Lp(a) was 127 mg/dL and LDL was 40 mg/dL and he had been on evolocumab and rosuvastatin. The corrected LDL accounts for the cholesterol contained in Lp(a) using the formula LDLcorr =LDL- 0.3x Lp(a) mass. Thus, his LDLcorr was zero. He underwent genetic testing with GBinsight which demonstrated he had Apo(a) short isoforms, which is associated with elevated Lp(a). He was also heterozygous for an extremely rare ABCG8 variant p.Gly212Glu (rs1318623110) which has not been previously reported. This variant is predicted to be deleterious and Gly212 is highly conserved across vertebrates. Mutations in ABCG5 or ABCG8 can be associated with increased LDL and risk for CAD. In addition, elevation of Lp(a) can be associated with 3 to 4 folds increased risk. This novel mutation, along with the elevated Lp(a) may help to explain his FH phenocopy presentation. Results We hypothesized that his premature disease progression may have been secondary to inflammation, atherogenesis or prothrombosis associated with Lp(a) with the added potentially deleterious effect of the ABCG8 mutation. His clinical course with his first MI at age 24 mimics that of FH, thus making him a phenocopy. We enrolled him in the randomized Lp(a)HORIZON trial evaluating the effect of pelacarsen in lowering Lp(a). However, he subsequently had an episode of unstable angina requiring additional intervention. Conclusions When assessing patients with suspected FH, mutations in genes other than LDLR, APOB, and PCSK9 should be evaluated. In this case, a mutation in ABCG8 gene along with Apo(a) short form isoforms may help explain the patient's disease progression and FH phenocopy presentation. In addition, ezetimibe may be beneficial in patients with ABCG5/8 mutation. His marked disease progression despite having a LDLcorr of zero highlights the importance of considering factors other than just LDL. Nothing to disclose.

Abstract 13685: Combined Genetic Defects of Sitosterolemia and Familial Hypercholesterolemia

Background: Familial hypercholesterolemia (FH) is an inherited disorder caused by mutations in the genes in the LDL receptor pathway, including LDLR , PCSK9 , APOB, and ARH . Defective uptake of LDL by the liver increases plasma LDL-cholesterol (LDL-C) levels, leading to an increased risk of coronary heart disease. The differential diagnosis of FH includes sitosterolemia which is a rare autosomal recessive disorder caused by mutations in ABCG5 or ABCG8 . Case presentation: Here we present a rare case of sitosterolemia with a genetic diagnosis of FH. A 64-years-old woman, clinically diagnosed as FH with Achilles tendon thickness (&gt;18 mm on X-ray imaging), had been taking statins only intermittently due to muscle pains. The combinatorial therapy with ezetimibe decreased her plasma LDL-C levels to around 90-250 mg/dL with variable adherence to statins and dietary management. Further addition of a PCSK9 inhibitor (evolocumab) was largely ineffective with plasma LDL-C levels of 90-190 mg/dL. A systemic medical evaluation revealed persistent joint pains of unknown etiology and slightly decreased platelet count. These clinical features led us to suspect sitosterolemia. Biochemical and genetic analyses: Plasma concentration of sitosterol was elevated (16.8 mg/dL). Genetic testing was performed after obtaining informed consent. All procedures were subjected to approval by the human genome, gene analysis research ethics committee of the University of Tokyo. In addition to a heterozygous missense mutation in LDLR (p.Arg115His), a homozygous nonsense mutation in ABCG5 (p.Tyr188Ter) was identified. No rare variants were found in PCSK9 nor ABCG8 . Conclusions: To our knowledge, this is the first case of a combined genetic defect of sitosterolemia and FH. Atypical clinical manifestations such as large fluctuations of LDL-C levels, unresponsiveness to a PCSK9 inhibitor, arthralgia, and a reduced platelet count led to the genetic diagnosis. Our case is informative in the perspective of precision medicine, highlighting the importance of systemic medical evaluation to select patients who will be benefited more from genetic testing as well as the necessity of future drug development for this rare and intractable disease.

Whole exome sequencing for diagnosis of hereditary thrombocytopenia.

Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management.To evaluate the role of whole exome sequencing (WES) in these Pts.Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM).Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9.This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

Serum sitosterol level predicting ABCG5 or ABCG8 genetic mutations

BackgroundCurrently, serum concentrations of sitosterol above 10 μg/ml are considered to be one of the major diagnostic criteria of sitosterolemia. MethodsWe retrospectively investigated consecutive 206 Japanese dyslipidemic subjects (mean age = 46 yr, male n = 94) with the assessments of serum sitosterol level and the presence of ABCG5 or ABCG8 genetic mutations in our institute since 2009–2018. We divided the subjects into 3 groups based on the number of pathogenic mutations in ABCG5 or ABCG8 genes. We compared serum lipids and serum sitosterol among those groups, and tried to validate the cutoff value discriminating patients of sitosterolemia with double mutations from others. ResultsWe identified 8 individuals with sitosterolemia with double mutations (affected), 26 individuals with a single mutation (carrier), and 172 individuals without any mutations (wildtype control). Serum sitosterol level of patients with sitosterolemia with double mutations (affected) exhibited significantly higher than those of any other groups (45.2. vs. 7.9 μg/ml, p = 2.5 × 10-2, 45.2 vs. 3.1 μg/ml, p = 1.8 × 10-2). Under these conditions, a cutoff value of sitosterol 10 μg/ml could discriminate the patients with sitosterolemia with double mutations in ABCG5 or ABCG8 gene from no mutation carrier (wildtype control) perfectly, although 6 heterozygous mutation carries exhibited sitosterol level greater than 10 μg/ml. Receiver-operating characteristic (ROC) analysis predicting double mutation status showed that the best cut-off value was 14.81 μg/ml. ConclusionWe suggest a cutoff value of sitosterol 15 μg/ml that shows higher positive predictive value than 10 μg/ml.

Compound heterozygous mutations in ABCG5 or ABCG8 causing Chinese familial Sitosterolemia.

Sitosterolemia (STSL), also known as phytosterolemia, is a rare autosomal recessive hereditary disease caused by mutations in the ABCG5 or ABCG8 genes. The disease is a result of disorders in lipoprotein metabolism, and is characterized by tendinous and tuberous xanthomas, elevated plasma cholesterol and phytosterol levels, and thrombocytopenia and hemolytic anemia in several patients. The manifestations of STSL are diverse and can easily be misdiagnosed. In recent years, cases of this disease in children have been reported in succession. There is therefore a need for clinicians to improve identification of STSL and perform early intervention. We evaluated four children with STSL caused by genetic mutations in ABCG5 or ABCG8, as well as their family members, by analyzing their clinical characteristics and performing Trio-whole exome sequencing. The biological consequences of the mutations were analyzed using various bioinformatics software. We also analyzed the consequences of a mutation commonly observed in STSL patients on the structure of the protein involved. We identified five previously unreported pathogenic mutations of different phenotypes of STSL: ABCG5 NM_022436:c.1337G>A; ABCG8 NM_022437:c.965-1G>A, c.323-1G>C, c.1418C>G and c.1534G>A. We also report the structural changes brought about by a mutation common in STSL patients, as well as the possible consequences of these changes. Our findings further broaden the genotypic and phenotypic profiles of the onset of STSL in the pediatric population and provide information for the diagnosis and treatment of this disease.

Beneficial effect of ezetimibe-atorvastatin combination therapy in patients with a mutation in ABCG5 or ABCG8 gene

BackgroundUse of ezetimibe on top of statin therapy has been shown to be effective to reduce LDL cholesterol level in hypercholesterolemic patients. However, little is known regarding the individual variety of the effectiveness of ezetimibe. We hypothesized that hypercholesterolemic patients with a mutation in ABCG5 or ABCG8 gene exhibit better response to ezetimibe than those without, based on the fact that ezetimibe is hyper-effective for in patients with sitosterolemia caused by ABCG5 or ABCG8 genetic mutations.MethodsElectronical medical record were reviewed in a total of 321 hypercholesterolemic patients (baseline LDL cholesterol = 192 ± 46 mg/dl) prescribed ezetimibe 10 mg daily on top of atorvastatin 10 mg daily who had undergone genetic analysis of ABCG5 or ABCG8 gene in our institute since 2006 to 2017. Pathogenicity of the variants were determined using standard variant filtering schema, including minor allele frequency, in silico annotation tools. Patients were divided into 2 groups based on the presence of ABCG5 or ABCG8 mutation. We compared the percent reduction of LDL cholesterol as well as the achieved LDL cholesterol levels between these 2 groups.ResultsWe found 26 (8%) individuals who exhibit deleterious mutations in ABCG5 or ABCG8 gene. Baseline characteristics under the atorvastatin 10 mg therapy were comparable in age, gender, and LDL cholesterol level between 2 groups. Under these conditions, percent reduction of LDL cholesterol in mutation positive group was significantly larger than that of mutation negative group (28 ± 16% vs. 39 ± 21%, p < 0.05). As a result, the achieved LDL cholesterol level in mutation positive group was significantly lower than that of mutation negative group (87 ± 29 mg/dl vs. 72 ± 26% mg/dl, p < 0.05).ConclusionThese results suggest that ezetimibe-atorvastatin combination therapy might be more beneficial in hypercholesterolemic patients with a mutation in ABCG5 or ABCG8 gene.

P6201Beneficial effect of ezetimibe-atorvastatin combination therapy in patients with a mutation in ABCG5 or ABCG8 gene

Abstract Background Use of ezetimibe on top of statin therapy has been shown to be effective to reduce LDL cholesterol level in hypercholesterolemic patients. However, little is known regarding the individual variety of the effectiveness of ezetimibe. We hypothesized that hypercholesterolemic patients with a mutation in ABCG5 or ABCG8 gene exhibit better response to ezetimibe that those without, based on the fact that ezetimibe is hyper-effective for in patients with sitosterolemia caused by ABCG5 or ABCG8 genetic mutations. Methods Electronical medical record were reviewed in a total of 321 hypercholesterolemic patients (baseline LDL cholesterol = 192±46 mg/dl) prescribed ezetimibe 10 mg daily on top of atorvastatin 10mg daily who had undergone genetic analysis of ABCG5 or ABCG8 gene in our institute since 2006 to 2017. Pathogenicity of the variants were determined using standard variant filtering schema, including minor allele frequency, in silico annotation tools. Patients were divided into 2 groups based on the presence of ABCG5 or ABCG8 mutation. We compared the percent reduction of LDL cholesterol as well as the achieved LDL cholesterol levels between these 2 groups. Results We found 26 (8%) individuals who exhibit deleterious mutations in ABCG5 or ABCG8 gene. Baseline characteristics under the atorvastatin 10mg therapy were comparable in age, gender, and LDL cholesterol level between 2 groups. Under these conditions, percent reduction of LDL cholesterol in mutation positive group was significantly larger than that of mutation negative group (28±16% vs. 39±21%, p&lt;0.05). As a result, the achieved LDL cholesterol level in mutation positive group was significantly lower than that of mutation negative group (87±29 mg/dl vs. 72±26% mg/dl, p&lt;0.05). Conclusion These results suggest that ezetimibe-atorvastatin combination therapy might be more beneficial in hypercholesterolemic patients with a mutation in ABCG5 or ABCG8 gene.

P6202Serum sitosterol levels and ABCG5 and ABCG8 genetic mutations among Japanese dyslipidemic subjects

Abstract Background Sitosterolemia, which is caused by ABCG5 or ABCG8 genetic mutations, is involved with marked increased concentrations of cholesterol as well as non-cholesterol plant sterols including sitosterol. Assessments of serum sitosterol level is critical to discriminate this disease from familial hypercholesterolemia (FH), which exhibits similar phenotypes except for elevated sitosterol levels. However, no prior data exist regarding the optimal cutoff value of serum sitosterol predicting the presence of sitosterolemia with double pathogenic mutations in ABCG5 or ABCG8 genes. Methods We retrospectively investigated consecutive 253 Japanese dyslipidemic subjects (mean age = 47.02 yr, male = 48.6%) with the assessments of serum sitosterol level as well as the presence of ABCG5 or ABCG8 genetic mutations in our institute since 2009 to 2018. Exome lesions of those 2 genes were sequenced, and pathogenicity of the identified genetic variants were determined using standard variant filtering schema, including minor allele frequency, in silico annotation tools. We divided the subjects into 3 groups based on the number of pathogenic mutations in ABCG5 or ABCG8 genes. We compared serum lipid values, including LDL cholesterol as well as serum sitosterol among those groups, and tried to determine the optimal cutoff value discriminating patients of sitosterolemia with double mutations from others. Results We identified 11 individuals with sitosterolemia with double mutations, 27 individuals with a single mutation, and 215 individuals without any mutations. Distribution of serum sitosterol exhibited skewed to the right (Figure). There was no significant correlation between serum sitosterol and other lipids, including LDL cholesterol. As expected, serum sitosterol level of patients with sitosterolemia exhibited significantly higher than those of any other groups (52.0 vs. 7.9 μg/ml, p=2.22×10–6, 52.0 vs. 2.8 μg/ml, p=2.275×10–8). 15 μg/ml was the optimal cut-off value, the sensitivity and specificity of which were 100%, and 99.6%, respectively. Histgram of serum sitosterol Conclusion We suggest the cutoff sitosterol value of 15 μg/ml predicting the presence of sitosterolemia with double pathogenic ABCG5 or ABCG8 genetic mutations.

Recurrent tendosynovitis as a rare manifestation of a lipid disorder

A 33-year-old female had suffered from spontaneously recurrent bursitis and tendosynovitis/enthesitis of the patellar and Achilles tendons for about 10 years. The episodes of immobilization increased. Ultrasound imaging of the swollen and painful tendons showed chronic inflammation with neoangiogenesis within the tendons and hypoechoic lesions. Clinical and laboratory tests did not provide evidence for a rheumatic disease. Low density lipoprotein cholesterol was elevated. Biopsies of skin lesions did not confirm the suspicion of cutaneous xanthomas. Genetic testing for familial hypercholesterolemia was negative. Campesterol and sitosterol were elevated 7- to 12-fold and 20- to 38-fold over the upper limit of normal on two occasions. There was no relevant mutation in ABCG5. In ABCG8, we identified a missense mutation c.1267G>A in exon 9 changing glutamic acid 423 into lysine within the transmembrane domain, and an insertion of adenine (c.1487insA) leading to a frameshift and a premature stop codon (Ile497Aspfs*105). The patient had no clinical evidence of premature atherosclerosis. Therapeutic approaches with nonsteroidal antirheumatic drugs, prednisone, statins, and ezetimibe accompanied by a diet poor in plant sterols led to a relief of symptoms. This case report shows that tendon xanthoma along with tendosynovitis, especially on extensor areas, is suspicious for hypercholesterolemia as the underlying cause. The absence of atherosclerotic plaques in the abdominal aorta and in the carotid arteries on ultrasound may suggest that phytosterolemia is not necessarily accompanied by premature vascular disease.

Sitosterolemia, Hypercholesterolemia, and Coronary Artery Disease.

Sitosterolemia is a rare inherited disease characterized by increased levels of plant sterols, such as sitosterol. The cause of this disease is ATP-binding cassette (ABC) subfamily G member 5 or member 8 (ABCG5 or ABCG8, respectively) gene mutations. Recent advances in genetics have revealed that the prevalence of subjects with deleterious mutations in ABCG5 and/or ABCG8 genes could be more than 1 in ∼200,000 individuals among the general population. Furthermore, accumulated evidence, including infantile cases exhibiting progression/regression of systemic xanthomas associated with LDL cholesterol levels, have shown that the elevation of LDL cholesterol seems to be the major cause of development of atherosclerosis and not the elevation of sitosterol. Regarding therapies, LDL apheresis, as well as proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, could be useful for sitosterolemia, in addition to ezetimibe and/or colestimide. In this study, we provide the current understanding and future perspectives of sitosterolemia, which is currently considered an extremely rare disorder but is expected to be much more prevalent in clinical settings.

First case of sitosterolemia caused by double heterozygous mutations in ABCG5 and ABCG8 genes

We present the first case of sitosterolemia caused by double heterozygous mutations in adenosine triphosphate-binding cassette subfamily G members 5 and 8 (ABCG5 and ABCG8) genes. A 1-year-old girl was admitted to Kanazawa University Hospital due to her hyper low-density lipoprotein (LDL)-cholesterolemia (453 mg/dL) as well as intertriginous xanthomas associated with breastfeeding. Initially, she was suspected as familial hypercholesterolemia (FH). However, her LDL cholesterol level significantly reduced after her weaning from breastfeeding. In addition, cascade screening did not show any evidence supporting dominant inheritance pattern as FH. Genetic analyses were performed using custom panel focusing on exome regions of 21 lipid-associated genes, including FH-causing genes (LDL receptor, proprotein convertase subtilisin/kexin type 9, apolipoprotein B), and ABCG5 and ABCG8 genes. In addition to a single deleterious mutation in ABCG5 gene (NM_022436.2:c.1166G>A or NP_071881.1:p.Arg389His), single deleterious mutation in ABCG8 gene (NM_022437.2:c.1285A>C or NP_071882.1:p.Met429Leu) was also identified. Segregations of those mutations from her parents were confirmed. Her serum sitosterol level was significantly elevated to 15.9 μg/mL, leading to her definite diagnosis as sitosterolemia. The ABCG5 and ABCG8 proteins form heterodimers and act as a complex. To the best of our knowledge, this is the first case exhibiting sitosterolemia caused by both ABCG5 and ABCG8 gene mutations.

Abstract 12291: Contribution of Rare Mutations in Mendelian Hypercholesterolemia Genes to Risk for Premature Coronary Artery Disease in the Population

Introduction: Low-density lipoprotein cholesterol (LDL-C) is a causal risk factor for coronary artery disease (CAD). Rare mutations in at least 6 genes lead to Mendelian forms of high or reduced LDL-C; three ( APOB, LDLR, PCSK9 ) act in a dominant pattern whereas three ( ABCG5, ABCG8, LDLRAP1 ) in a recessive pattern. We address to what extent rare mutations in Mendelian LDL-C genes contribute to early CAD risk in the population. Methods: We sequenced the exons of the 6 genes in 9,329 early CAD cases (myocardial infarction, angiographic CAD, or coronary revascularization in men≤50 and women≤60) and 10,245 controls from 9 studies using targeted and whole exome next-generation sequencing. We tested 3 sets: ‘Null alleles’ (nonsense, splice-site, or frameshift); ‘Deleterious (7/7)’ (null and missense annotated as damaging by 7 algorithms); and ‘Deleterious (6/7)’ (null and missense annotated as damaging by at least 6 algorithms). Given the rarity of deleterious mutations, we aggregated these mutations in each gene and tested for an excess or deficit in cases vs . controls. Results: Counts of mutations are provided in Table. Null mutations in LDLR , carried by 1:500 participants, confered a 8-fold increase in CAD risk (P=8х10 -7 ) whereas heterozygosity for a null mutation in ABCG5 (1:650 frequency) was associated with a 3-fold increased risk (P=5х10 -3 ). ‘Deleterious (7/7)’ mutations in LDLR , carried by 1:100 participants, confered a 4-fold increased risk (P=8х10 -17 ) whereas heterozygosity for a ‘Deleterious (7/7)’ mutation in ABCG5 (1:250 frequency) was associated with a 2-fold increased risk (P=2х10 -3 ). Heterozygous null allele carriers at LDLR and ABCG5 had increased LDL-C (P&lt;0.001). Conclusions: Of early CAD cases, 2-3% carry a rare, deleterious mutation at LDLR or ABCG5 associated with increased risk. Although previously reported to cause recessive sitosterolemia, we find that heterozygosity for a null allele at ABCG5 is associated with markedly higher early CAD risk.

A novel mutation of ABCG5 gene in a Turkish boy with phytosterolemia presenting with macrotrombocytopenia and stomatocytosis

Phytosterolemia is a rare autosomal recessive sterol storage disease caused by mutations in ABCG5 and ABCG8 genes. A 9-year-old Turkish boy who was presented with exclusively hematologic abnormalities had elevated plant sterol levels. Sequencing of ABCG5 and ABCG8 genes revealed a novel homozygous IVS10-1 G>T mutation in ABCG5 gene. Four of the 13 family members had xanthoma but they had neither hematologic abnormalities nor IVS10-1 G>T mutation. Ezetimibe therapy reduced plant sterol levels in association with marked clinical improvement. Plant sterol levels and ABCG5/ABCG8 genes should be analysed in patients with unexplained hemolytic anemia and macrothrombocytopenia.

Hepatobiliary transport in health and disease

Bile salts, cholesterol and phosphatidylcholine are secreted across the canalicular membrane of hepatocytes into bile by ATP-binding cassette (ABC) transporters. Secretion of bile salts by ABCB11 is essential for bile flow and for absorption of lipids and fat-soluble vitamins. ABCG5 and ABCG8 eliminate excess cholesterol and sterols from the body by secreting them into bile. There are two mechanisms to protect the canalicular membrane from solubilization by bile salts; ABCB4 secretes phosphatidylcholine into bile to form mixed micelles with bile salts, and ATP8B1 maintains the canalicular membrane in a liquid-ordered state. Three different forms of progressive familial intrahepatic cholestasis (PFIC) disorders, PFIC1, PFIC2 and PFIC3, are caused by mutations in ATP8B1, ABCB11 and ABCB4, respectively. Sitosterolemia is caused by mutations in ABCG5 and ABCG8. This article reviews the physiological roles of these canalicular transporters, and the pathophysiological processes and clinical features associated with their mutations.

Effect of a Liver X Receptor Agonist on Deposition and Lymphatic Absorption of Plant Sterols in Stroke-Prone Spontaneously Hypertensive Rats Having a Mutation in ATP-Binding Cassette Transporter G5

The effects were compared of T0901317, a liver X receptor agonist, on deposition in the liver and serum and lymphatic absorption of plant sterols in stroke-prone spontaneously hypertensive rats (SHRSPs) having a missense mutation in Abcg5, which codes for ATP-binding cassette transporter (ABC) G5, with those in Wistar rats. Both strains were pair-fed for 7 d with a 0.5% plant sterol diet with or without 5 mg/kg of body weight of T0901317. The deposition of plant sterols in the liver and serum was higher in SHRSPs than in Wistar rats. A significant reduction of plant sterol deposition was observed in Wistar rats, but not in SHRSPs when T0901317 was given. Both strains were then fed for 7 d with a control diet with or without T0901317. The lymphatic absorption of plant sterols was reduced to almost half the normal level by the T0901317 treatment. However, no difference in absorption was apparent between SHRSPs and Wistar rats regardless of the T0901317 treatment. These results suggest that the plant sterol deposition in SHRSPs was not necessarily caused by the increased absorption of plant sterols.

ATP-binding cassette G5/G8 deficiency causes hypertriglyceridemia by affecting multiple metabolic pathways

Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/−) and ABCG5/G8-deficient (G5/G8−/−) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/− and G5/G8−/− mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8−/− mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/− (1.3-fold) and G5/G8−/− mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/− (1.8-fold) and G5/G8−/− mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/− and G5/G8−/− than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-3H] oleate, was 5.8-fold higher in G5/G8−/− than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8−/− than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.

P285 FIRST DEMONSTRATION OF THE LOCALIZATION OF HEPATIC apoB100- AND INTESTINAL apoB48-CONTAINING LIPOPROTEIN PARTICLES IN HUMAN ATHEROSCLEROTIC PLAQUES USING DUAL-STAINING IMMUNOFLUORESCENCE

Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/−) and ABCG5/G8-deficient (G5/G8−/−) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/− and G5/G8−/− mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8−/− mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/− (1.3-fold) and G5/G8−/− mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/− (1.8-fold) and G5/G8−/− mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/− and G5/G8−/− than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-3H] oleate, was 5.8-fold higher in G5/G8−/− than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8−/− than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.