Apolipoprotein A-I Mutations and Cardiovascular Disease Ernst J. Schaefer, MD Distinguished University Professor Human Nutrition Research Center on Aging at Tufts University, Tufts University School of Medicine, Boston, MA & Chief Medical Officer, Boston Heart Diagnostics, Framingham, MA May 26th, 2015 ISA 2015, Amsterdam, the Netherlands Decreased HDL-C and ApoA-I Framingham Offspring Study

• • • Diabetes • Male gender • Sedentary lifestyle • Smoking

Schaefer EJ, Lamon-Fava S, Ordovas JM, Cohn SD, Schaefer MM, Castelli WP, Wilson PWF J Res. 35:871-882, 1994. Human Apolipoprotein (Apo) Metabolism Synthetic Sites Catabolic Sites

FFA Chylomicron LPL, HL, LCAT Chylomicron 5 hours ApoB-48 Remnant other Apos 4 ApoB-48, ApoE 6 Lipids 1 8 Kidney Intestine HDL 12 mg/kg/d ApoA-I 3 other Apos Scavenger Liver Lipids Cells 9 2

VLDL 12 mg//kg/d LDL ApoB-100 ApoB-100 Peripheral other Apos LPL, HL, 5 Lipids 3.5 days Cells Lipids LCAT 7 FFA 4 hours

Schaefer & Levy New Engl J Med 1985; 312:1300, Cohn JS et al J Clin Invest 1990: 85:804, Schaefer EJ. Am J Clin Nutr. 2002;75:191 Disorders in Families with Premature CHD

• Familial Lipoprotein (a) Excess: 19%

• Familial (High TG, Low HDL): 15%

• Familial Combined : 14% (high LDL-C &TG, usually low HDL-C)

• Familial : 4%

• Familial : 1%

Genest JJ, Martin-Munley S, McNamara JR, Ordovas JM, Jenner J, Myers R, Wilson PW, Schaefer EJ. Circulation 85:2025-2033, 1992. (use of 10th and 90th percentile cutpoints from Framingham ApoA-I-Containing HDL Subpopulation Profiles of a Control and a CHD Patient pre  pre [nm]

17.0

9.51 8.16

7.10 4.66

Asztalos et al. Arterioscler Thromb Vasc Biol. 2003;23:847-852; 2004; 24:2181-2187, 2005; 25:2185-2191, HATS Trial, Framingham Offspring Study, and the Veterans Affairs HDL Intervention Trial Differences in HDL Particles in Male CHD Cases vs Controls in the Framingham Offspring Study

Apo A-I Concentration % Difference Controls (n=1277) Cases (n=169)

17 mg/dl - 39%* (16% of total) 40 mg/dl - 9% (37% of total) 38 mg/dl + 29%* (36% of total) 12 mg/dl + 16%* (11% of total) “For each 1 mg/dl apoA-I increase in alpha 1 HDL - 26% reduction in CHD risk Asztalos BF, Cupples LA, Demissie S, Horvath KV, Cox CE, Batista MC, Schaefer EJ. Arterioscler Thromb Vasc Biol 2004; 24:2181-2187. Differences in HDL Particles in Female CHD Cases vs Controls

Apo A-I Concentration % Difference Controls (n=126) Cases (n=256) 27 mg/dl - 49%* (17% of total)

51 mg/dl - 25% (33% of total) 41 mg/dl - 8% (26% of total) 17 mg/dl + 41%* (11% of total) * P< 0.05 versus controls Lamon-Fava S et al Arterioscler Thromb Vasc Biol 2008;28:575-9. HDL Particles and Recurrent CHD Events in VA HIT (1097 without vs 398 with recurrent events)

Apo A-I Concentration % Difference Recurrent Dis. 9.1 mg/dl - 12%** (54% of normal) 31.9 mg/dl - 7% (80% of normal) 40.5 mg/dl - 3%* (107% of normal) 12.1mg/dl + 9%* (100% of normal) “In CHD patients with low HDL those with lower α-1 HDL on trial were significantly more likely to have recurrent events.” Asztalos BF, Collins D, Cupples LA, Demissie S, Horvath KV, Bloomfield HE, Robins SJ, Schaefer EJ. Arterioscler Thromb Vasc Biol 2005; 25:2185-2191. Effects of 80 mg/dl vs. 40 mg/dl on HDL Particles vs Baseline in STELLAR (n=306)

Atorvastatin Rosuvastatin

+ 12%* + 24%*

+ 4% + 13*

-7% - 6%

- 40%* - 39%* Asztalos BF, LeMaulf F, Dallal GE, Stein E, Jones PH, Horvath KV, McTaggert F, Schaefer EJ. Am J Cardiol 2007; 99: 681-685. Effects of Simvastatin/Niacin on HDL Particles in CHD Cases in HATS (n=123)

Apo A-I Concentration % Difference

9 mg/dl + 115%*

30 mg/dl + 27%

45 mg/dl - 17%*

15 mg/dl - 39%* “The increase in apoA-I in large alpha 1 HDL was significantly related (p<0.01) to lack of progression or regression of coronary artery stenosis.” Asztalos BF, Batista M, Horvath KV, Cox CE, Dallal GE, Morse JS, Brown GB, Schaefer EJ. Arterioscler Thromb Vasc Biol 2003;23:847-852. Low HDL in Population

 3 candidate genes (ABCA1, APOA1, and LCAT) sequenced in subjects < 5th percentile of HDL-C vs. > 95th percentile of HDL-C.

 NS variants were significantly more common (12% versus 2%) in individuals with low HDL-C (n=284) than in those with high HDL-C (n=236).

 9.9% ABCA1, 2.2% LCAT, and 0.2% APOA1

Cohen JC et al Science 2004; 305:869-72. Apolipoprotein A-I Variants in the General Population

 Sequenced APOA1 gene in 10,330 population-based participants in the Copenhagen City Heart Study.  Only 0.27% of individuals in the general population were heterozygous for NS variants which were associated with substantial reductions in apoA-I (up to 39 mg/dL) and/or HDL (up to 0.9 mmol/L).  0.41% of the population were heterozygous for variants predisposing to amyloidosis.  NS variants were associated with a hazard ratio of 1.72 (CI, 1.09-2.70, p<0.01) for myocardial infarction (MI), largely driven by A164S, a variant usually not associated with low apoA-I or HDL cholesterol levels.

Haase CL, Frikke-Schmidt R, Nordestgaard BG, Tybjaerg-Hansen A, PLoS Genetics 2012;8(11):e1003063 ApoA-I Mutations and Amyloidosis

 In hereditary amyloidosis apoA-I mutations cause its proteolysis, and the depositions of 9-11 kd N terminal fragments as fibrils in the kidney, liver, and heart, damaging these organs.  All amyloidogenic apoA-I mutations cluster in residue segments 26-107 and 154 -178, and can destabilize lipid free and HDL bound apoA-I.

 ApoA-I Iowa (G26R) is among the best characterized of these variants.

Gursky O et al. Biochemistry 2012;51:10-18. Marked HDL Deficiency

HDL-C Levels < 20 mg/dL in the absence of liver disease, TG > 1000 mg/dL, or testosterone use Usually due to mutations at either the APOAI, ABCAI (), or LCAT gene loci They can be distinguished based on a standard lipid profile, HDL particle analysis, and corneal examination. Parameters in Marked Deficiency

HDL- Direct LDL- ApoAI in Parameter C/ApoAI C/TG HDL (mg/dL) (mg/dL) Particles AI/CIII/AIV Deficiency ** 2/0 126/56 ND AI/CIII Deficiency ** 2/0 135/62 ND AI Deficiency** 2/0 126/56 ND Tangier Disease (ABCA1)** 3/2.0 52/159 Preβ-1 Preβ-1, α-4, LCAT Deficiency 7/52 0/139 larger HDL

Fish Eye Disease* 6/36 157/158 Preβ-1, α-4 Preβ-1, α-4, Controls 55/150 120/100 3,2, and 1

Premature CVD, ** prior to age 40, ** prior to age 60 , * prior to age 70 years, Schaefer EJ et al Curr. Opin. Lipidol 2010; 21:289-97. Apolipoprotein A-I Immunoblotting in Various Disorders of HDL Metabolism

Schaefer EJ, Santos RD, Asztalos BF. Curr Opin Lipidol 2010;21:289-97. Arcus Juvenilis, Tendinous , Heterozygous Familial Hypercholesterolemia Atypical Arcus Juvenilis, Xanthomas, ApoA-I Deficiency, CVD age 39 years,homozygous nonsense codon -2 in APOAI gene. Santos R et al. J Lipid Res 2008;49: 349-357. Female with homozygous Tangier disease, developed neuropathy at age 25 years, CHD at age 45 years, died from CHD at age 58 years, at autopsy severe diffuse CAD. Her diffuse corneal opacification was best seen on slit lamp examination; ABCA1 mutation Asn to His at residue 1740 in 2nd transmembrane domain. Brousseau et al J Lipid Res 2000;41:433-441 52 year old male with diffuse corneal opacification & atypical arcus juvenilis, renal insufficiency, & anemia, LCAT mutation homozygous Met to Arg at residue 293, received LCAT enzyme replacement at NIH for 10 weeks (Dr. Remaley), stabilized renal disease & improved anemia, but shortage of enzyme, Rx stopped, and now on dialysis, Roshan et al J Clin Lipidol 2011;5:493-99. HDL Particles Before and After LCAT Enzyme Replacement in LCAT Deficient Patient Treated at NIH by Dr. Alan Remaley and Colleagues Atypical arcus juvenilis in a healthy 23 year woman and positive family history of premature CHD, compound heterozygous mutation P34L and T137M in LCAT gene, data consistent with Fish Eye Disease. Dimick et al. J Clin Lipidol 2014; 8:223-230. 22 Confidential – Boston Heart Diagnostics Familial Apolipoprotein A-I Deficiency

 Patients who lack AI/CIII/AIV (deletion of gene complex) have marked HDL deficiency, low TG levels, normal LDL-C levels, premature CHD (female age 43 died of CHD after bypass) (Schaefer et al Arteriosclerosis, 1982, JLR 1985, Ordovas et al JBC 1989)  Patients who lack AI/CIII (DNA rearrangement) have marked HDL, low TG levels, normal LDL-C levels, premature CHD (two sisters required bypass at 29 and 30 years of age, since then have died of CHD), planar xanthomas, and normal fat absorption (Norum et al NEJM, 1982, Karathanasis PNAS 1987)  Patients who lack AI only have marked HDL deficiency, normal TG levels, and premature CHD: 1) Homozygous nonsense mutation, codon 84, CHD in Japanese female in her 50s. (Matsunaga et al PNAS, 1991) 2) 4 homozygotes in kindred by Q[-2]X mutation in Canada, 3 had CHD in their 30s, one was in her 20s, no CHD. (Ng et al JCI, 1994) 3) Two brothers with severe CHD at 38 and 39 requiring bypass, tubo-eruptive and planar xanthomas, normal LDL-C and TG; Q[-2]X mutation, (Santos et al, JLR 2008) Apolipoprotein A-I Variants  ApoA-I - 243 residues with repeats of amphipathic alpha helices. The N and C terminal helices (1,9, and 10) are essential for lipid binding. The C terminal helix (helix 10) is involved in ABCA1 binding and cell mediated cholesterol efflux. The central helices 5, 6 and 7 are critical for LCAT activation.  There are now 82 apoA-I mutations listed in the Human Gene Mutation Database (www.hgmd.cf.ac.uk).  The mutations are from -2 to 216. There are 5 nonsense mutations, 58 missense mutations, 14 deletions, 4 insertions, 1 splice site mutation, and 3 complex rearrangements.

Schaefer EJ, Santos RD, Asztalos BF. Curr Opin Lipidol 2010;21:289-97. Apolipoprotein A-I Variants and LCAT Activity

 Mutations associated with abnormal LCAT activation are located within helices 5, 6, and 7, corresponding to amino acids 121 to 187.  Three heterozygous apoA-I missense mutations (Leu141Arg)Pisa, (Arg160Leu)Oslo, and (Pro165Arg) have been associated with low HDL-C have been associated with decreased LCAT activity. None of the affected patients had evidence of CHD.  However patients who were compound heterozygotes for apoA-I (Leu141Arg)Pisa, and an apoA-I null allele have been reported to have very low HDL-C and apoA-I levels that were 3% of control value. All of these subjects had marked corneal opacification and three of the four compound heterozygotes had significant premature CHD.

Schaefer EJ, Santos RD, Asztalos BF. Curr Opin Lipidol 2010;21:289-97. ApoA-I Nashua  A 61-year-old male with significant coronary heart disease since age 42 years, corneal arcus, and marked high- density lipoprotein (HDL) deficiency (HDL-C 1 mg/dL, apoA-I 23 mg/dL, and only preβ-1 and α-2 HDL particles).  Novel heterozygous inframe insertion mutation with duplication of nucleotides 1535 through 1552 inserted at position 1553, causing a new amino acid glycine at residue 157 and a duplication of amino acids alanine, arginine, alanine, histidine, and leucine at residues 158–162.  This novel apoA-I mutation appears to result in the formation of apoA-I that has abnormal lipid binding properties, resulting in probable impaired reverse cholesterol transport , and premature CHD. Lee EY, Klementowicz PY, Hegele RA, Asztalos BF, Schaefer EJ. J. Clin Lipidol 2013; 7: 169–173. ApoA-I Mytilene  The proband was a 68-year-old male with premature coronary heart disease since age 54 years, corneal arcus, and an HDL-C 14 mg/dL, apoA-I 57 mg/dL, and lack of very α-1 HDL. Two other family members also had the same pattern.  The affected kindred members were noted to have an

apoA-I truncation (apoA-I Mytilene) due to a heterozygous nonsense mutation (c.718C > T, p.Gln216*) resulting in a truncated apoA-I containing only 215 amino acids.  Kinetic studies indicated that the proband had an apoA-I production rate (PR) that was 40% of normal, and cellular cholesterol efflux capacity that was 65% of normal.

Anthanont P, Polisecki B, Asztalos BF, Diffenderfer MP, Barrett HR, Millar JR, Billheimer B, Cuchel M, Rader DJ, Schaefer EJ. 2014: 235: 470-476. ApoA-I Truncations

 10 kindreds with apoA-I truncations reported.  3 kindreds had hereditary amyloidosis.  3 kindreds affected with heterozygous apoA-I truncations, low HDL-C and apoA-I, with two kindreds (including ours) having premature CHD.  5 kindreds affected with homozygous apoA-I truncations, very low HDL-C and apoA-I, as well as LCAT deficiency and corneal opacification. Two of these had evidence of premature CHD. ApoA-I Boston  Proband was a 67 year woman with HDL deficiency (HDL-C 8 mg/dL, apoA-I 75 mg/dL, and lack of very large α-1 HDL. Her two sons had similar values.  All affected family members had a novel heterozygous apoA-I missense mutation apoA-I

Boston. (Arg149Ser)  Affected subjects were noted to have decreased serum cellular cholesterol efflux and LCAT activity. No premature CHD was noted in the kindred. Anthanont P, Asztalos BF, Polisecki E, Zachariah B, Schaefer EJ. J Clin Lipidol 2015 (in press). Conclusions  Patients who have undetectable plasma apoA-I and marked HDL deficiency usually develop CHD prior to age 40 years.  Patients with amino acid substitutions can have a variable picture, with those having an apoA-I variant associated with decreased LCAT activation often not having premature CHD (usually heterozygotes for mutations in helices 5 – 7).  All known human apoA-I amyloidogenic mutations are clustered in two residue segments, 26-107 and 154-178, and promote apoA-I proteolysis, and deposits in tissues.  Patients with homozygous or heterozygous apoA-I mutations affecting N or C terminal amino acids often have abnormal lipid binding and may have premature CHD. Conclusions

 The only lipid disorder associated with earlier premature CHD than homozygous apoA-I deficiency (undetectable apoA-I) is homozygous familial hypercholesterolemia (FH).  The pattern of coronary atherosclerosis is different in these two disorders.  In homozygous FH the patients develop ostial disease and aortic valve cholesterol deposition, while in homozygous apoA-I deficiency they develop more typical diffuse coronary atherosclerosis. Sprecher D et al. Am J Cardiol. 1984; 54:20-30, Schaefer EJ et al. J Lipid Res. 1985; 26:1089-101, Santos R et al. Atherosclerosis. 2008;197:910-5, J Lipid Res. 2008; 49:349-57.