Genetics of Familial Hypercholesterolemia 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: A709, Research Building 2011/03

Learning Objectives l. 掌握家族性高胆固醇血症致病基因的有 关知识。 2. 了解LDL在细胞内的相关转运和代谢途 径。

Required Reading Thompson &Thompson Genetics in Medicine, 7th Ed （双语版，2009） ● P275-280; ● Clinical Case Studies-14 Familial Hypercholesterolemia

Cardiovascular Disorders Heart disease is the leading cause of death worldwide, and it accounts for approximately 25% of all deaths in the US. The most common underlying cause of heart disease is coronary artery disease (CAD), which is caused by atherosclerosis. A number of risk factors for CAD have been identified, including obesity, cigarette smoking, hypertension, elevated cholesterol level, and positive family history (usually defined as having one or more affected first-degree relatives).

Risk for CAD? The risk is higher: if there are more affected relatives. if the affected relative is female (the less commonly affected sex) rather than male. if the age of onset in the affected relative is early (before 55 years of age).

What part do genes play in the familial clustering of CAD?

LDLR gene → FH (OMIM: 143890 ) An important advance was the isolation and cloning of the gene (1984) that encodes the low-density lipoprotein (LDL) receptor. Heterozygosity for a mutation in LDLR (19p13.2) roughly doubles LDL cholesterol levels and is seen in approximately 1 in 500 persons ---- familial hypercholesterolemia (FH) ---- accounting for approximately 5% of myocardial infarctions (MIs) in persons younger than 60 years.

The 4 Genes Associated with FH Mutant Gene Product Pattern of Inheritance Prevalence Effect of Disease- Causing Mutations Typical LDL Cholesterol Level (Normal Adults: ~120 mg/dL) LDL receptor AD (19p13.2) HTZs: 1/500 HMZs: 1/106 Loss of function HTZs: 350 HMZs: 700 Apoprotein B-100 (2p24) HTZs: 1/1000* HMZs: 1/106* HTZs: 270 HMZs: 320 ARH adaptor Pr. AR (1p36-p35) Very rare† HMZs: 470 PCSK9 protease (1p34.1-p32 ) Very rare Gain of function HTZs: 225

The 4 proteins associated with FH

The 4 proteins associated with FH The LDL receptor binds apoprotein B-100. Mutations in the LDL receptor binding domain of apoprotein B-100 impair LDL binding to its receptor, reducing the removal of LDL cholesterol from the circulation. Clustering of the LDL receptor–apoprotein B-100 complex in clathrin-coated pits requires the ARH adaptor protein, which links the receptor to the endocytic machinery of the coated pit. Homozygous mutations in the ARH protein impair the internalization of the LDL:LDL receptor complex, thereby impairing LDL clearance. PCSK9 protease activity leads to degradation of the LDL receptor.

Clinical Synopsis of FH INHERITANCE: AD HEAD AND NECK: Eyes: (1) Corneal arcus; (2) Xanthelasma. CARDIOVASCULAR: Heart: CAD presenting after age 30 years in heterozygotes, in childhood in homozygotes. SKIN, NAILS, HAIR: Skin: (1)Tendinous xanthomas presenting after age 20 years in heterozygotes, during first 4 years of life in homozygotes; (2)Planar xanthomas in homozygotes. LABORATORY ABNORMALITIES: Hypercholesterolemia, 350-550 mg/dL in heterozygotes, 650-1000 mg/dL in homozygotes. (normal: ~300-400 mg/dL) MISCELLANEOUS: Incidence, 1/500 heterozygotes, 1/106 homozygotes. MOLECULAR BASIS: Caused by mutations in LDLR.

Arcus lipoides

Xanthomas (fatty deposit)

Xanthomas (fatty deposit)

Xanthomas (fatty deposit)

FH

LDL receptor: a membrane-bound 160-kD Pr. of 839 AAs

The process of receptor-mediated endocytosis

The process of receptor-mediated endocytosis 1, LDL receptors, which are glycoproteins, are synthesized in the ER of the cell. 2, They pass through the Golgi apparatus to the cell surface, where part of the receptor protrudes outside the cell. 3, The circulating LDL particle is bound by the LDL receptor and localized in cell-surface depressions called coated pits (so named because they are coated with a protein called clathrin). 4, The coated pit invaginates, bringing the LDL particle inside the cell. 5, Once inside the cell, the LDL particle is separated from the receptor, taken into a lysosome, and broken down into its constituents by lysosomal enzymes. 6, The LDL receptor is recirculated to the cell surface to bind another LDL particle. Each LDL receptor goes through this cycle approximately once every 10’, even if it is not occupied by an LDL particle. 7, Free cholesterol is released from the lysosome for incorporation into cell membranes or metabolism into bile acids or steroids. Excess cholesterol can be stored in the cell as a cholesterol ester or removed from the cell by association with HDL. 8, As cholesterol levels in the cell rise, cellular cholesterol synthesis is reduced by inhibition of the rate-limiting enzyme, HMG-CoA reductase. 9, Rising cholesterol levels also increase the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT), an enzyme that modifies cholesterol for storage as cholesterol esters. 10, In addition, the number of LDL receptors is decreased by lowering the transcription rate of the LDL receptor gene itself. This decreases cholesterol uptake.

LDLR mutations: 5 broad classes I: result in no detectable protein product. Thus, heterozygotes would produce only half the normal number of LDL receptors. II: result in production of the LDL receptor, but it is altered to the extent that it cannot leave the endoplasmic reticulum. It is eventually degraded. III: produce an LDL receptor that is capable of migrating to the cell surface but is incapable of normal binding to LDL. IV: which are comparatively rare, produce receptors that are normal except that they do not migrate specifically to coated pits and thus cannot carry LDL into the cell. V: produce an LDL receptor that cannot disassociate from the LDL particle after entry into the cell. The receptor cannot return to the cell surface and is degraded.

LDL receptor and the 6 classes of mutations that alter its function

The cell biology and biochemical role of the LDL receptor and the six classes of mutations that alter its function After synthesis in the endoplasmic reticulum (ER), the receptor is transported to the Golgi apparatus and subsequently to the cell surface. Normal receptors are localized to clathrin-coated pits, which invaginate, creating coated vesicles and then endosomes, the precursors of lysosomes. Normally, intracellular accumulation of free cholesterol is prevented because the increase in free cholesterol (A) decreases the formation of LDL receptors, (B) reduces de novo cholesterol synthesis, and (C) increases the storage of cholesteryl esters. The biochemical phenotype of each class of mutant is discussed in the text. ACAT, acyl coenzyme A:cholesterol acyltransferase; HMG CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The structure of the LDL receptor gene showing its 5 domains and the effect on the receptor of mutations in these domains that lead to FH

Each class of mutations reduces the number of effective LDL receptors, resulting in decreased LDL uptake and hence elevated levels of circulating cholesterol

Common PCSK9 Variants Associated with Low LDL Cholesterol Levels Sequence Variant Population Frequency of Heterozygotes Mean Reduction in LDL Cholesterol Impact on Incidence of CAD Tyr142Stop or Cys679Stop Arg46Leu African Americans: 2.6% Whites: 3.2% 28% (38 mg/dL) 15% (20 mg/dL) 90% reduction 50% reduction

The Nobel Prize in Medicine 1985: Brown MS & Goldstein JL "for their discoveries concerning the regulation of cholesterol metabolism"

FH approximately 75% of men with FH developed CAD, and 50% had a fatal MI, by age 60 years. The corresponding percentages for women were lower (45% and 15%, respectively). Most homozygotes experience MIs before 20 years of age, and an MI at 18 months of age has been reported. Without treatment, most FH homozygotes die before the age of 30 years.

Age- and Sex-specific rates (%) of CAD and death in FH heterozygotes Males Females Age CAD Death 30 5 — 40 20-24 0-3 50 45-51 25 12-20 2 60 75-85 45-57 15 70 100 80 75

Therapy for FH heterozygotes Dietary reduction of cholesterol has only modest effects. The administration of bile-acid absorbing resins, such as cholestyramine. However, the decrease in intracellular cholesterol also stimulates cholesterol synthesis by liver cells, so the overall reduction in plasma LDL is only about 15% to 20%. This treatment is much more effective when combined with one of the statin drugs (e.g., lovastatin, pravastatin), which reduce cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Serum cholesterol levels in FH heterozygotes can often be reduced to approximately normal levels.

Therapy for FH homozygotes Homozygotes have few or no LDL receptors. Liver transplants, which provide hepatocytes that have normal LDL receptors, have been successful in some cases. Plasma exchange, carried out every 1 to 2 weeks, in combination with drug therapy, can reduce cholesterol levels by about 50%. However, this therapy is difficult to continue for long periods. Somatic cell gene therapy, in which hepatocytes carrying normal LDL receptor genes are introduced into the portal circulation, is now being tested.

Suggested readings Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia. Hum Mol Genet. 2010;19(22):4313-8. Genetics of coronary artery disease. Annu Rev Genomics Hum Genet. 2010;11: 91-108.