CLINICAL RESEARCH CLINICAL RESEARCH www.nature.com/clinicalpractice/cardio APOA5 genetic variants are markers for classic hyperlipoproteinemia phenotypes and hypertriglyceridemia 1 1 1 2 2 1 1 Jian Wang , Matthew R Ban , Brooke A Kennedy , Sonia Anand , Salim Yusuf , Murray W Huff , Rebecca L Pollex and Robert A Hegele1* SUMMARY INTRODUCTION Hypertriglyceridemia is a common biochemical Background Several known candidate gene variants are useful markers for diagnosing hyperlipoproteinemia. In an attempt to identify phenotype that is observed in up to 5% of adults. other useful variants, we evaluated the association of two common A plasma triglyceride concentration above APOA5 single-nucleotide polymorphisms across the range of classic 1.7 mmol/l is a defining component of the meta­ 1 hyperlipoproteinemia phenotypes. bolic syndrome and is associated with several comorbidities, including increased risk of cardio­ Methods We assessed plasma lipoprotein profiles and APOA5 S19W and vascular disease2 and pancreatitis.3,4 Factors, –1131T>C genotypes in 678 adults from a single tertiary referral lipid such as an imbalance between caloric intake and clinic and in 373 normolipidemic controls matched for age and sex, all of expenditure, excessive alcohol intake, diabetes, European ancestry. and use of certain medications, are associated Results We observed significant stepwise relationships between APOA5 with hypertriglyceridemia; however, genetic minor allele carrier frequencies and plasma triglyceride quartiles. The factors are also important.5,6 odds ratios for hyperlipoproteinemia types 2B, 3, 4 and 5 in APOA5 S19W Complex traits, such as plasma triglyceride carriers were 3.11 (95% CI 1.63−5.95), 4.76 (2.25−10.1), 2.89 (1.17−7.18) levels, usually do not follow Mendelian patterns of and 6.16 (3.66−10.3), respectively. For APOA5 –1131T>C carriers, the inheritance because multiple genes contribute to odds ratios for these hyperlipoproteinemia subtypes were 2.23 (95% the phenotypes.7 In very rare instances, however, CI 1.21−4.08), 3.18 (1.55−6.52), 3.95 (1.85−8.45) and 4.24 (2.64−6.81), relevant mutations in single genes have been respectively. The overall odds ratio for the presence of either allele in lipid found. Children with hyperchylomicronemia clinic patients was 2.58 (95% CI 1.89−3.52). (WHO International Statistical Classification Conclusions A high proportion of patients with four classic of Diseases, 10th edition, classification E78.3; hyperlipoproteinemia phenotypes are carriers of either the APOA5 S19W Fredrickson hyperlipoproteinemia type 1; or –1131T>C variant or both. These two variants are robust genetic Mendelian Inheritance in Man, 12th edition, classifi­ biomarkers of a range of clinical hyperlipoproteinemia phenotypes linked cation 238600) often have homozygous muta­ by hypertriglyceridemia. tions in LPL encoding lipoprotein lipase (LPL) or KEYWORDS APOA5, complex trait, DNA variant, hyperlipoproteinemia, APOC2 encoding apolipoprotein (apo) C­II, an triglyceride LPL cofactor. Indeed, five of the six classic WHO or Fredrickson hyperlipoproteinemia pheno­ types include varying degrees of elevated fasting plasma triglyceride in their definitions (Table 1). The exception is familial hypercholesterolemia (FH) (International Statistical Classification of Diseases, 10th edition, classification 78.0; hyper­ lipoproteinemia type 2A; Mendelian Inheritance 1Vascular Biology Research Group, Robarts Research Institute and Schulich School of Medicine in Man, 12th edition, classification 143890), and Dentistry, University of Western Ontario, London, ON N6A 5K8, Canada 2 which is most frequently caused by mutations in Population Health Research Institute, McMaster University, Hamilton Health Sciences, 8 Hamilton, ON L8L 2X2, Canada LDLR encoding the LDL receptor. The genetic basis of hyperlipoproteinemia types 1 and 2A Correspondence have, therefore, been identified. Most cases of *Blackburn Cardiovascular Genetics Laboratory, Robarts Research Institute, 406-100 Perth Drive, Box 5015, London, ON N6A 5K8, Canada hyperlipoproteinemia types 2B, 3, 4 and 5 are Tel: +1 519 663 3461 only partially characterized at the molecular [email protected] genetic level, although it is known that type 3 requires homozygosity for the APOE E2 isoform Received 21 February 2008 Accepted 21July 2008 Published online 9 September 2008 www.nature.com/clinicalpractice as necessary for expression of the phenotype, doi:10.1038/ncpcardio1326 but this factor is not sufficient alone.2,9 Given 730 NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE NOVEMBER 2008 VOL 5 NO 11 CLINICAL RESEARCH CLINICAL RESEARCH www.nature.com/clinicalpractice/cardio Table 1 Summary of classic hyperlipoproteinemia phenotypes. WHO ICD-10 Frederickson HLP phenotype MIM Lipid Lipoprotein Genetic characteristics code number levels levels E78.3 HLP type 1 238600 TG CM Monogenic, autosomal recessive due to Familial chylomicronemia mutant LPL or APOC2. Primarily pediatric and young adults E78.0 HLP type 2A 143890 TC LDL Monogenic, heterozygous form due to mutant Familial hypercholesterolemia LDLR, APOB or PCSK9; Homozygous form due to mutant LDLR or ARH E78.4 HLP type 2B 144250 TC, TG VLDL, Polygenic, multiple etiologies, some cases due Combined hyperlipoproteinemia LDL to USF1, APOB or LPL E78.2 HLP type 3 107741 TC, TG IDL Polygenic, APOE E2/E2 homozygosity or mutant Dysbetalipoproteinemia APOE necessary but not sufficient E78.1 HLP type 4 144600 TG VLDL Polygenic, no specific genes yet identified Primary hypertriglyceridemia and 145750 or replicated E78.3 HLP type 5 144650 TC, TG VLDL, Polygenic, mutant LPL, APOC2 and APOA5 Mixed hyperlipidemia CM in ~10% of cases Abbreviations: APOA5, gene encoding apolipoprotein A-V; APOB, gene encoding apolipoprotein B; APOC2, gene encoding apolipoprotein C-II; APOE, gene encoding apolipoprotein E; ARH, gene encoding autosomal recessive hypercholesterolemia protein; CM, chylomicrons; HLP, hyperlipoproteinemia; ICD, International Statistical Classification of Diseases, 10th Edition; IDL, intermediate-density lipoprotein; LDLR, gene encoding LDL receptor; LPL, lipoprotein lipase; LPL, gene encoding LPL; MIM, Mendelian Inheritance in Man; PCSK9, gene encoding proprotein convertase subtilisin/kexin type 9; TC, total cholesterol; TG, triglyceride; USF1, gene encoding upstream stimulatory factor 1. that hypertriglyceridemia is common, and that METHODS most classic hyperlipoproteinemia phenotypes Participants defined in part by elevated triglyceride levels have We studied 678 consecutive, unrelated white no identified genetic basis, a DNA marker that is patients from a tertiary referral lipid clinic (age consistently associated with hypertriglyceridemia range 18−84 years). Patients underwent a complete might help both in understanding pathogenesis medical examination and provided a history, and in diagnosis. and basic clinical, biochemical and demographic The apolipoprotein apo A­V is encoded by variables were recorded. Normolipidemic adult APOA5 and is a key apolipoprotein whose controls were selected from the European sub­ physiological role has been demonstrated group of the Study of Health Assessment and Risk in studies showing elevated triglyceride levels in in Ethnic groups (SHARE), a survey of cardio­ knockout mice,10 elevated triglyceride levels vascular risk factors in Canadian subpopulations,16 in probands with rare loss­of­function muta­ and from population­based controls from Ontario tions in APOA511,12 and associations of single­ who self­reported good health. By use of a validated nucleotide polymorphism (SNP) genotypes or sampling strategy,16 households of white ethnicity haplotypes with raised plasma triglyceride concen­ within the same geographic area from which the trations.13,14 This apolipoprotein appears to patients were referred were randomly selected and play a focal part in the hydrolysis of triglyceride­ mailed an introductory letter. Written contact was rich lipoproteins by increasing the activity followed by up to 12 telephone calls inviting the of LPL; rare mutations of apo A­V can cause individual with the earliest date of birth from familial chylomicronemia.11,12,15 The SNPs the household to participate. All study partici­ S19W and –1131T>C in APOA5 are relatively pants provided informed consent for DNA analy­ common (i.e. 5−10% allele frequency) in most sis (University of Western Ontario Institutional populations; they are defining variants of unique Review Board protocol number #07920E). APOA5 haplotypes, are associated with in vitro dysfunction and are consistently associated with Biochemical determinations and elevated plasma triglyceride levels.12 We there­ hyperlipoproteinemia phenotype fore evaluated APOA5 S19W and –1131T>C Plasma lipoprotein profiles were determined as genotypes for association with elevated plasma previously described for lipid clinic patients17 triglyceride levels and hyperlipoproteinemia and for normal controls.16 Lipid clinic patients phenotypes in patients from a lipid clinic. were classified as having FH based on the presence NOVEMBER 2008 VOL 5 NO 11 WANG ET AL. NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE 731 CLINICAL RESEARCH CLINICAL RESEARCH www.nature.com/clinicalpractice/cardio of characteristics that definitely met clinical present to allow the custom probe to be designed and biochemical diagnostic criteria,8 including (Applied Biosystems). The custom probe uses the demonstration of heterozygosity for a disease­ primers 5'­CCC TGC GAG TGG AGT TCA­3' and causing mutation.18,19 Participants