Journal of Clinical Lipidology (2015) 9, 390–395
Case Studies
Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux Pimjai Anthanont, MD, Bela F. Asztalos, PhD, Eliana Polisecki, PhD, Benoy Zachariah, MD, Ernst J. Schaefer, MD* Cardiovascular Nutrition Laboratory, Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA (Drs Anthanont, Asztalos, and Schaefer); Boston Heart Diagnostics, Framingham, MA, USA (Drs Asztalos, Polisecki, and Schaefer); Tufts University School of Medicine, Boston, MA, USA (Drs Asztalos and Schaefer); and Steward Health Good Samaritan Cardiology Group, Brockton, MA, USA (Dr Zachariah) KEYWORDS: HDL deficiency; Apolipoprotein A-I; Genetic mutation; Cellular cholesterol efflux; LCAT activity; CETP activity
Abstract: We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C.A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large a-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P , .05). The percentage of HDL cholesterol in the esterified form was also significantly (P , .05) reduced to 52% of control values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I–mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P , .05). lecithin-cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P , .05). The data indicate that this novel apoA-I missense is associated with markedly decreased levels of HDL cholesterol and very large a-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity. Ó 2015 National Lipid Association. All rights reserved.
Source of financial support: This research was supported by Boston Heart Diagnostics, Framingham, Massachusetts. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the US Department of Agriculture or the National Institutes of Health. * Corresponding author. Cardiovascular Nutrition Laboratory, Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail address: [email protected] Submitted July 11, 2014. Accepted for publication February 18, 2015.
1933-2874/Ó 2015 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2015.02.005
Introduction Markedly decreased plasma or serum levels of highdensity lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I), in the absence of severe hypertriglyceridemia or liver disease, can be due to mutations in the genes for apoA-I, adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1), and lecithin-cholesterol acyltransferase
Anthanont et al
ApoA-I variant, cholesterol efflux, and LCAT activity
(LCAT).1 Patients with these mutations can be differentiated by the measurement of plasma or serum apoA-I concentrations and the determination of apoA-I in individual HDL particles by 2-dimensional gel electrophoresis followed by immunoblotting using antisera specific for apoA-I.1 ApoA-I, the major protein constituent of HDL, has been shown to activate LCAT, bind lipids, promote cellular cholesterol efflux, and form mature HDL.2 Based on our review of the literature, 82 apoA-I mutations have been reported in human subjects.1,3,4 Most apoA-I mutations are missense mutations resulting in a change in the amino acid sequence of the full-length protein. Approximately 40% of apoA-I missense mutations are associated with low HDL-C and apoA-I concentrations, whereas the remaining mutations are associated with either normal HDL-C with or without hereditary amyloidosis. ApoA-I is a protein containing 243 amino acids, and has 8 repeats of 22 amino acids and 2 repeats of 11 amino acids.5 Mutations associated with abnormal LCAT activation are located within repeats 5, 6, and 7, corresponding to amino acids 121 to 186.5 Three heterozygous apoA-I missense mutations (Leu141Arg)Pisa, (Arg160Leu)Oslo, and (Pro165Arg) have been associated with low HDL-C, decreased LCAT activity, and decreased cellular cholesterol efflux.6–9 None of the affected patients had evidence of coronary heart disease (CHD). However, patients who were compound heterozygotes for apoA-I (Leu141Arg)Pisa and an apoA-I null allele have been reported to have undetectable HDL-C levels and apoA-I levels that were 3% of control value.10 All these subjects had marked corneal opacification, and 3 of the 4 compound heterozygotes had significant premature CHD.9,10 In this study, we report a kindred affected with HDL deficiency and a novel heterozygous apoA-I missense mutation (Arg149Ser), which would be predicted to affect LCAT activation. To assess the effect of the mutation on HDL functionality, we also measured serum cellular cholesterol efflux capacity, LCAT activity, Cholesteryl ester tranfer protein (CETP) activity, and the percentage of cholesterol that was esterified in plasma and in the HDL fraction.
Materials, methods, and results Report of kindred The proband was a 67-year-old female (Fig. 1, II-6) who presented to our lipid and heart disease prevention clinic with a history of very low HDL-C for at least 20 years. Her HDL-C, triglycerides (TGs), and low-density lipoprotein cholesterol levels off medication had ranged from 6 to 22, 65 to 350, and 95 to 130 mg/dL, respectively, between the ages of 43 and 67 years. Four years before being seen in our clinic, she developed chest discomfort and was evaluated by echocardiography and nuclear stress testing. These tests revealed a normal left ventricular ejection fraction and normal myocardial perfusion, respectively. Since that time she has had no further symptoms and no evidence
391
Figure 1 Pedigree of the proband’s family. Arrow indicates the proband. The numbers below the symbols indicate age at the time of this inquiry or at the time of death. The causes of death are described under the symbols. CHD, coronary heart disease.
of heart disease, stroke, or peripheral vascular disease. She did have a history of treated hypertension and hypothyroidism, but no history of diabetes or smoking. She had a prior history of statin-induced myalgia, but has been able to tolerate low-dose pravastatin (5 mg orally every other day). She also now takes ezetimibe 10 mg/d, antihypertensive medications, and levothyroxine. On these medications, her low-density lipoprotein cholesterol value was reduced from 122 to 75 mg/dL. Her most recent physical examination revealed: a body mass index of 35.7 kg/m2, a blood pressure of 160/80 mm Hg, a heart rate of 70 beats/min, with a normal heart and lung examination, and neurologic function, and normal pulses. She had no evidence of hepatosplenomegaly, xanthomas, orange tonsils, arcus senilis, or corneal opacification. Her most recent plasma lipid values on the aforementioned medications are shown in Table 1. She also had normal liver and thyroid function tests, normal creatine kinase levels, but her creatinine levels were mildly elevated. More optimal blood pressure control was recommended for implementation by her referring physician. Her spouse was sampled and found to have normal lipids. The proband’s kindred is shown in the Figure 1. The proband’s father (I-3) was of Irish origin. He had a myocardial infarction at the age of 50 years and died at the age of 54 years of CHD. One of his brothers (I-2) died at the age of 75 years of CHD. The proband’s mother (I-4) had a history of smoking and hypertension, and died at the age of 73 years of lung cancer. No lipid levels were available for these subjects. The proband’s sister (II-4) also had a history of low HDL-C (4–10 mg/dL) and high TGs (200–450 mg/dL), as did one of her daughters (III-5; HDL-C, 4–7 mg/dL and TG, 650–1150 mg/dL). The proband’s sister had a history of obesity and diabetes. However, neither of these subjects had any evidence of heart disease and did not want to be sampled for this study. One brother aged 74 years, had normal lipids, and was in good health.
392 Table 1
Journal of Clinical Lipidology, Vol 9, No 3, June 2015 Lipid, lipoprotein, and apolipoprotein values in cases and controls
Parameter
Proband (II-6)
Age (y) 67 TC (mg/dL) 176 Triglycerides (mg/dL) 215 LDL-C (mg/dL) 122 HDL-C (mg/dL) 8 ApoA-I (mg/dL) 74.7 ApoB (mg/dL) 87 Lp(a) (mg/dL) 11.4 Apo A-I concentrations in HDL subpopulations a-1 (very large) 4.1 a-2 (large) 29.6 a-3 (medium) 13.1 a-4 (small) 16.2 Preb-1 (very small) 6.3
Offspring 1 (III-7)
Offspring 2 (III-8)
Mean 6 SD (%) affected (n 5 3)
Mean 6 SD controls (n 5 20)
39 137 220 95 3 35.8 94 12.0 (mg/dL) 2.9 15.6 4.2 4.9 3.3
35 146 160 100 7 66.8 83 14.15
47.0 6 17.4 153.0 6 20.4 198.0 6 33.3* 106.0 6 14.4 6.0 6 2.6* 59.1 6 20.6* 88.0 6 5.6 12.5 6 3.2
(83.6) (185.0) (101.9) (10.0) (35.0) (107.3) (54.5)
58.0 183.2 107.4 103.6 60.1 169.3 81.7 23.1
6 6 6 6 6 6 6 6
15.1 37.4 37.2 33.0 15.7 32.5 22.6 27.3
3.4 6 0.6* 22.5 6 7.0* 10.1 6 5.1 12.9 6 7.0 4.9 6 1.5
(15.7) (53.6) (76.5) (119.4) (92.5)
21.6 42.0 13.2 10.8 5.3
6 6 6 6 6
4.7 3.5 2.6 2.5 1.7
3.4 22.4 13.1 17.8 5.1
Apo, apolipoprotein; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Lp(a), lipoprotein(a); SD, standard deviation; TC, total cholesterol. Values in parentheses indicate the percentage of the mean normal value based on 10 normal male and 10 normal female controls. *P , .05 as compared with controls.
The proband had 2 sons (III-7 and III-8), both of whom had a history of low HDL-C (less than 20 mg/dL), with no history of CHD or smoking. Subject III-7 was sampled while on atorvastatin 20 mg/d (placed on this medication because of prior significant hyperlipidemia with total serum cholesterol and TG values that were both .250 mg/dL). He also had significant obesity with a body mass index of 41.2 kg/m2. Subject III-8 was sampled off medication and had a body mass index of 25.6 kg/m2. Both these subjects had normal liver, kidney, and thyroid function studies. There were 10 normal male and 10 normal female control subjects used for this study. All subjects were sampled after an overnight fast and were not on medications known to affect lipid metabolism. All females were premenopausal. Their mean age and body mass index was 34.5 years and 25.1 kg/m2, respectively.
Laboratory analysis Lipid, apolipoprotein, and HDL particle profiles Laboratory results on blood obtained after an overnight fast were analyzed at Boston Heart Diagnostics, Framingham, Massachusetts, as previously described.3,4,11 The concentrations of lipids, lipoproteins, apolipoproteins, and HDL particle profiles in the family members and control subjects are shown in Table 1. Compared with controls, the proband and her affected sons had significant increases in serum TG levels that were 185% of control values (P , .05), and significant reductions in serum HDL-C and apoA-I concentrations that were 10% and 35% of control values, respectively (both P , .05). Statistical analysis was carried out using paired t test analysis comparing values in the 3 affected subjects with the 20 control subjects.
HDL particles were analyzed by 2-dimensional gel electrophoresis followed by apoA-I antibody immunoblotting. The concentration of apoA-I in mg/dL within each HDL subpopulation was calculated, as described previously.1,3,4,11,12 These results showed that the mean apoA-I concentrations in very large a-1, large a-2, medium a-3, small a-4, and very small preb-1 HDL particles in affected family members were 15.7%, 53.6%, 76.5%, 119.4%, and 92.5%, respectively, of values in controls. The reductions in the very large a-1 and large a-2 HDL particles were significant (P , .05) compared with controls. In contrast, the unaffected spouse of the proband had normal values for all HDL particle parameters. The percentage of total cholesterol in the esterified form and phospholipid levels were assessed in whole plasma, the non-HDL fraction, and the HDL fraction in affected subjects and controls as previously described in our laboratory at Tufts University.12–15 The mean percentages of total cholesterol in the esterified form in affected subjects in plasma, non-HDL, and HDL were 83%, 91%, and 52% of control values, respectively (only the latter value in HDL was significantly decreased, P , .05). CETP mass levels were measured as previously described in whole plasma, non-HDL, and HDL of affected subjects and controls, and values in affected subjects were 89%, 110%, and 49% of control values, respectively (only the latter value in HDL was significantly decreased, P , .05).15,16 The level of CETP and LCAT activity were measured using plasma and HDL as previously described and were found to be 92% and 71% of controls values (the latter being significant at P , .05).16 The mean levels of phospholipids in plasma, non-HDL, and HDL in affected subjects measured as previously described were 110%, 91%, and 15% of controls values, respectively (only the latter value
Anthanont et al Table 2
ApoA-I variant, cholesterol efflux, and LCAT activity
393
Cholesterol efflux capacity and LCAT activity in the proband’s family
Subject Proband (II-6) III-7 III-8 Mean 6 SD Controls (n 5 20)
Global cholesterol efflux (% per 4 h) (1cAMP) 5.96 4.25 5.76 5.32 6 0.93* (41.4%) 12.86 6 2.87
Cholesterol efflux mediated by (% per 4 h) ABCA1
SR-BI
Ex vivo LCAT activity (% esterified per 4 h)
2.58 2.21 2.30 2.36 6 0.19* (36.5%) 6.46 6 2.19
1.59 0.96 1.86 1.47 6 0.46* (41.0%) 3.59 6 0.86
12.68 12.08 11.72 12.16 6 0.48* (73.4%) 16.57 6 2.19
ABCA1, ATP-binding cassette transporter A1; Apo, apolipoprotein; cAMP, cyclic adenosine monophosphate; 1cAMP, cAMP-treated J774 cells; LCAT, lecithin-cholesterol acyltransferase; SD, standard deviation; SR-BI, scavenger receptor B1. Values in parentheses indicate percentage of values obtained using apoB-depleted control human serum from control subjects. *P , .05 as compared with controls.
in HDL was significantly decreased, P , .05).15 Plasma apoA-II levels were measured by immunoassay as previously described, and mean values in the affected subjects were significantly reduced (P , .05) at 45% of values in controls.15 DNA sequencing Because of our laboratory findings, DNA was isolated from white blood cells obtained from the proband, her 2 sons, and her spouse. The APOA1 gene was sequenced using Sanger sequencing methodology using our core sequencing facility. In this study, the protein sequence numbers refer to the mature apoA-I (243 amino acids), and the nucleotide numbers refer to the reference sequence NM_000039.1 (nucleotide 1 is the A of the ATG-translation initiation codon). DNA sequencing revealed that the proband (II-6) and her 2 sons (III-7 and III-8) were found to be heterozygous for a C to A substitution at nucleotide 517 (c.517C.A). This mutation creates an amino acid substitution changing arginine to serine at residue 149 (p.Arg149Ser). The proband’s spouse (II-7) had a normal APOA1 sequence. In addition, the LCAT gene was sequenced in the proband and was found to be normal. Assessment of cellular cholesterol efflux capacity and ex vivo LCAT activity Cholesterol efflux capacities of serum apoB-depleted samples (3 affected subjects, 1 nonaffected subject, and 20 control subjects) were determined, as described previously, under contract in the Vascular Strategies Laboratory, Plymouth Meeting, Pennsylvania.17 Global and ABCA1mediated cholesterol efflux were measured using mouse J774 macrophages in the presence and absence of cyclic adenosine monophosphate (cAMP). Scavenger receptor B type I (SR-BI)–mediated efflux was measured using rat Fu5AH hepatoma cells. For all assays, cells were labeled by incubation with [3H]-cholesterol and the acetyl-coenzyme A aceyltransferase inhibitor Sandoz 58-035 for 24 hours. Cells were then incubated overnight in 0.2% bovine serum albumin. Results were obtained as previously described.17,18 Ex vivo LCAT activity, the proportion of [3H]-cholesterol
released from cAMP-treated J774 cells to serum HDL during the 4-hour period that was esterified by serum LCAT in the extracellular medium was measured. The cholesterol efflux capacity and ex vivo LCAT activity of affected subjects and controls are shown in Table 2. The mean global cholesterol efflux, ABCA1mediated efflux, and SR-B1–mediated efflux values of affected family members were 53.0%, 46.2%, and 52.1%, respectively, of control values (all significantly reduced, P , .05). In addition, affected family member had mean serum LCAT activity that was 73.4% of control values (also significantly reduced, P , .05).
Discussion The proband in this kindred had severe HDL deficiency, mild hypertriglyceridemia, and detectable large a-1 and a-2 HDL particles by 2-dimensional gel electrophoresis, although the levels of these particles were significantly reduced. The most likely possibility therefore was an apoAI variant because patients with homozygous Tangier disease due to ABCA1 mutations generally have only preb-1 HDL present in plasma, whereas those with homozygous LCAT deficiency generally have only have preb-1 and a-4 HDL present in plasma.1 DNA sequencing revealed a heterozygous missense mutation of APOA1 (c.517C.A, p.Arg149Ser), which we have designated as apoA-IBoston. The proband and her 2 sons had severe HDL deficiency, and all were heterozygous for apoA-IBoston. In addition, the proband’s LCAT gene was found to be normal. The heterozygous form of this mutation has resulted in a dominant negative phenotype because the apoA-I levels in the affected subjects were ,50% of control values. A similar situation has been observed with 7 other apoA-I variants: apoA-IFin, apoA-IZavalla, apoA-IMallorca, apoAISeattle, and apoA-IMilano.2,5–10,19 ApoA-IFin and apoAIZavalla are missense mutations at residue 159 in helix 6, whereas apoA-ISeattle and apoA-IMallorca are deletion mutations within helix 6 and 7, respectively. ApoA-IMilano is characterized by the substitution of arginine by cysteine
394 at residue 173, resulting in the formation of a disulfidelinked apoA-IMilano homodimer, as well as a heterodimer with apoA-II. The proposed mechanism of this group implies that the oligomeric (dimer or tetrameric) apoA-I, constituted both by wild type and mutant, would have a reduced capacity to activate the enzyme LCAT. Moreover, the lack of efficient cholesterol esterification causes the formation of lipid-poor HDL that is rapidly catabolized. ApoA-I plays a role in LCAT activation. The function of LCAT is to transfer a fatty acid from lecithin or phosphatidylcholine to free cholesterol on the surface of lipoproteins to form cholesteryl ester, which migrate into the core of HDL, resulting in the formation of mature, spherical medium and large a migrating HDL particles. It has been established by Sorci-Thomas and Thomas5 as well as other investigators that the central a-helical region (residues 121–186) of apoA-I are involved in LCAT activation.2 It has been reported that 3 arginine residues in this region (Arg-149, Arg-153, and Arg-160) are critical for the ability of apoA-I to activate LCAT.18 In this kindred, affected family members had mean apoA-II, apoA-I, HDL phospholipid, and HDL-C levels that were all significantly reduced at 45%, 35%, 15%, and 10%, respectively of control values. Moreover the percentage of HDL-C as cholesteryl ester and CETP mass in HDL were both significantly reduced to 52% and 49% of control values, and the levels of apoA-I in very large a-1 and large a-2 HDL particles were also significantly reduced at 16% and 54% of control values, respectively. All affected subjects in the kindred were found to be heterozygous for an apoA-I mutation causing the replacement of an arginine with a serine at residue 149 (Arg149Ser). Based on prior studies, it would be predicted that this amino acid substitution would have an effect on the ability of apoA-I to activate LCAT. HDL functionality studies documented that the mean global cholesterol efflux, ABCA1-mediated efflux, and SR-B1–mediated efflux values of affected family members were significantly reduced to 53%, 46%, and 52%, respectively, of control values. These decreases are commensurate with decreases in HDL particle mass. Moreover, mean ex vivo apoB-depleted serum LCAT activity was significantly reduced at 73.4% of control values. This assay reflects the proportion of labeled cholesterol released from cAMP-treated J774 cells to serum HDL during a 4-hour period that was esterified by serum LCAT. The overall finding in this kindred with no evidence of premature CHD in the proband or her affected younger sons are consistent with observations in 3 other kindreds with heterozygous apoA-I missense mutations (Leu141Arg)Pisa, (Arg160Leu)Oslo, and (Pro165Arg), which were associated with low HDL-C, decreased LCAT activity, decreased cellular cholesterol efflux, and no evidence of premature CHD. It should also be noted that neither we nor others have observed premature CHD in patients with familial LCAT deficiency despite very low HDL-C levels.12,14
Journal of Clinical Lipidology, Vol 9, No 3, June 2015 However, the proband’s father did die of premature CHD at the age of 54 years. An important function of HDL particles is the promotion of global cellular cholesterol efflux, which has been associated with CHD risk.20,21 Cellular cholesterol efflux can be assessed in a global fashion, as well as by also examining ABCA1-mediated and SR-BI–mediated efflux, as was done in this kindred. The primary HDL particle serving as an acceptor of cholesterol and phospholipids via the ABCA1 transporter is very small lipid-poor preb-1 HDL.18 In contrast, the preferred HDL particle for bidirectional cholesterol flux mediated via the SR-BI transporter is very large lipid-rich a-1 HDL.18 In our view, the SR-BI–mediated cholesterol efflux in this kindred was decreased because of a significant decrease in very large a-1 HDL particles. We postulate that the reduction in the ABCA1-mediated cholesterol efflux in this kindred was because of both a modest decrease in preb-1 HDL and a reduction in LCAT activity and cholesterol esterification. To our knowledge, there is no prior report of this mutation in humans.
Financial disclosures The authors have no conflicts of interest to disclose. P.A. was a postdoctoral research fellow from Thammasat University, Thailand. B.F.A., E.P., and E.J.S. are employees and have ownership interest in Boston Heart Diagnostics, Framingham, Massachusetts. This research was supported by the US Department of Agriculture Research Service Contract 53-3K-06 (E.J.S.) and Project Grant P50 HL083813-01 from the National Institutes of Health (E.J.S.).
References 1. Schaefer EJ, Santos RD, Asztalos BF. Marked HDL deficiency and premature coronary heart disease. Curr Opin Lipidol. 2010;21: 289–297. 2. Frank PG, Marcel YL. Apolipoprotein A-I: structure-function relationships. J Lipid Res. 2000;41:853–872. 3. Lee EY, Klementowicz PT, Hegele RA, et al. HDL deficiency due to a new insertion mutation (ApoA-INashua) and review of the literature. J Clin Lipidol. 2013;7:169–173. 4. Anthanont P, Polisecki E, Asztalos BF, et al. A novel ApoA-I truncation (ApoA-IMytilene) associated with decreased ApoA-I production. Atherosclerosis. 2014;235:470–476. 5. Sorci-Thomas MG, Thomas MJ. The effects of altered apolipoprotein A-I structure on plasma HDL concentration. Trends Cardiovasc Med. 2002;12:121–128. 6. von Eckardstein A, Funke H, Henke A, et al. Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I. J Clin Invest. 1989;84: 1722–1730. 7. Leren TP, Bakken KS, Daum U, et al. Heterozygosity for apolipoprotein A-I(R160L) Oslo is associated with low levels of high density lipoprotein cholesterol and HDL-subclass LpA-I/A-II but normal levels of HDL-subclass LpA-I. J Lipid Res. 1997;38:121–131.
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8. Daum U, Leren TP, Langer C, et al. Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I (P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers. J Lipid Res. 1999;40:486–494. 9. Miccoli R, Zhu Y, Daum U, et al. A natural apolipoprotein A-I variant, apoA-I (L141R)Pisa, interferes with the formation of alpha-high density lipoproteins (HDL), but not with the formation of pre beta 1-HDL and influences efflux of cholesterol into plasma. J Lipid Res. 1997;38:1242–1253. 10. Miccoli R, Bertolotto A, Navalesi R, et al. Compound heterozygosity for a structural apolipoprotein A-I variant, apo A-I (L141R) Pisa, and an apolipoprotein A-I null allele in patients with absence of HDL cholesterol, corneal opacifications, and coronary heart disease. Circulation. 1996;94:1622–1628. 11. Asztalos BF, Cupples LA, Demissie S, et al. High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants in the Framingham Offspring Study. Arterioscler Thromb Vasc Biol. 2004;24:2181–2187. 12. Roshan B, Ganda OP, Desilva R, et al. Homozygous lecithin:cholesterol acyltransferase (LCAT) deficiency due to a new loss of function mutation and review of the literature. J Clin Lipidol. 2011;5: 493–499. 13. Dimick SM, Sallee B, Asztalos BF, et al. A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy. J Clin Lipidol. 2014;8:223–230.
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14. Asztalos BF, Schaefer EJ, Horvath KV, et al. Role of LCAT in HDL remodeling: investigation of LCAT deficiency states. J Lipid Res. 2007;48:592–599. 15. Asztalos BF, Horvath KV, Kajinami K, et al. Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency. J Lipid Res. 2004;45:448–455. 16. Asztalos BF, Swarbrick MM, Schaefer EJ, et al. Effects of weight loss, induced by gastric bypass, on HDL remodeling in obese women. J Lipid Res. 2010;51:2405–2412. 17. de la Llera-Moya M, Drazul-Schrader D, Asztalos BF, et al. The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages. Arterioscler Thromb Vasc Biol. 2010; 30:796–801. 18. Asztalos BF, de la Llera-Moya M, Dallal GE, et al. Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux. J Lipid Res. 2005;46:2246–2253. 19. Roosbeek S, Vanloo B, Duverger N, et al. Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase. J Lipid Res. 2001;42:31–40. 20. Khera AV, Cuchel M, de la Llera-Moya M, et al. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis. N Engl J Med. 2011;364:127–135. 21. Rohatgi A, Khera A, Berry JD, et al. HDL cholesterol efflux capacity and incident cardiovascular events. N Engl J Med. 2014;371:2383–2393.
Case Studies
Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux Pimjai Anthanont, MD, Bela F. Asztalos, PhD, Eliana Polisecki, PhD, Benoy Zachariah, MD, Ernst J. Schaefer, MD* Cardiovascular Nutrition Laboratory, Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA (Drs Anthanont, Asztalos, and Schaefer); Boston Heart Diagnostics, Framingham, MA, USA (Drs Asztalos, Polisecki, and Schaefer); Tufts University School of Medicine, Boston, MA, USA (Drs Asztalos and Schaefer); and Steward Health Good Samaritan Cardiology Group, Brockton, MA, USA (Dr Zachariah) KEYWORDS: HDL deficiency; Apolipoprotein A-I; Genetic mutation; Cellular cholesterol efflux; LCAT activity; CETP activity
Abstract: We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C.A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large a-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P , .05). The percentage of HDL cholesterol in the esterified form was also significantly (P , .05) reduced to 52% of control values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I–mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P , .05). lecithin-cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P , .05). The data indicate that this novel apoA-I missense is associated with markedly decreased levels of HDL cholesterol and very large a-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity. Ó 2015 National Lipid Association. All rights reserved.
Source of financial support: This research was supported by Boston Heart Diagnostics, Framingham, Massachusetts. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the US Department of Agriculture or the National Institutes of Health. * Corresponding author. Cardiovascular Nutrition Laboratory, Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail address: [email protected] Submitted July 11, 2014. Accepted for publication February 18, 2015.
1933-2874/Ó 2015 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2015.02.005
Introduction Markedly decreased plasma or serum levels of highdensity lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I), in the absence of severe hypertriglyceridemia or liver disease, can be due to mutations in the genes for apoA-I, adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1), and lecithin-cholesterol acyltransferase
Anthanont et al
ApoA-I variant, cholesterol efflux, and LCAT activity
(LCAT).1 Patients with these mutations can be differentiated by the measurement of plasma or serum apoA-I concentrations and the determination of apoA-I in individual HDL particles by 2-dimensional gel electrophoresis followed by immunoblotting using antisera specific for apoA-I.1 ApoA-I, the major protein constituent of HDL, has been shown to activate LCAT, bind lipids, promote cellular cholesterol efflux, and form mature HDL.2 Based on our review of the literature, 82 apoA-I mutations have been reported in human subjects.1,3,4 Most apoA-I mutations are missense mutations resulting in a change in the amino acid sequence of the full-length protein. Approximately 40% of apoA-I missense mutations are associated with low HDL-C and apoA-I concentrations, whereas the remaining mutations are associated with either normal HDL-C with or without hereditary amyloidosis. ApoA-I is a protein containing 243 amino acids, and has 8 repeats of 22 amino acids and 2 repeats of 11 amino acids.5 Mutations associated with abnormal LCAT activation are located within repeats 5, 6, and 7, corresponding to amino acids 121 to 186.5 Three heterozygous apoA-I missense mutations (Leu141Arg)Pisa, (Arg160Leu)Oslo, and (Pro165Arg) have been associated with low HDL-C, decreased LCAT activity, and decreased cellular cholesterol efflux.6–9 None of the affected patients had evidence of coronary heart disease (CHD). However, patients who were compound heterozygotes for apoA-I (Leu141Arg)Pisa and an apoA-I null allele have been reported to have undetectable HDL-C levels and apoA-I levels that were 3% of control value.10 All these subjects had marked corneal opacification, and 3 of the 4 compound heterozygotes had significant premature CHD.9,10 In this study, we report a kindred affected with HDL deficiency and a novel heterozygous apoA-I missense mutation (Arg149Ser), which would be predicted to affect LCAT activation. To assess the effect of the mutation on HDL functionality, we also measured serum cellular cholesterol efflux capacity, LCAT activity, Cholesteryl ester tranfer protein (CETP) activity, and the percentage of cholesterol that was esterified in plasma and in the HDL fraction.
Materials, methods, and results Report of kindred The proband was a 67-year-old female (Fig. 1, II-6) who presented to our lipid and heart disease prevention clinic with a history of very low HDL-C for at least 20 years. Her HDL-C, triglycerides (TGs), and low-density lipoprotein cholesterol levels off medication had ranged from 6 to 22, 65 to 350, and 95 to 130 mg/dL, respectively, between the ages of 43 and 67 years. Four years before being seen in our clinic, she developed chest discomfort and was evaluated by echocardiography and nuclear stress testing. These tests revealed a normal left ventricular ejection fraction and normal myocardial perfusion, respectively. Since that time she has had no further symptoms and no evidence
391
Figure 1 Pedigree of the proband’s family. Arrow indicates the proband. The numbers below the symbols indicate age at the time of this inquiry or at the time of death. The causes of death are described under the symbols. CHD, coronary heart disease.
of heart disease, stroke, or peripheral vascular disease. She did have a history of treated hypertension and hypothyroidism, but no history of diabetes or smoking. She had a prior history of statin-induced myalgia, but has been able to tolerate low-dose pravastatin (5 mg orally every other day). She also now takes ezetimibe 10 mg/d, antihypertensive medications, and levothyroxine. On these medications, her low-density lipoprotein cholesterol value was reduced from 122 to 75 mg/dL. Her most recent physical examination revealed: a body mass index of 35.7 kg/m2, a blood pressure of 160/80 mm Hg, a heart rate of 70 beats/min, with a normal heart and lung examination, and neurologic function, and normal pulses. She had no evidence of hepatosplenomegaly, xanthomas, orange tonsils, arcus senilis, or corneal opacification. Her most recent plasma lipid values on the aforementioned medications are shown in Table 1. She also had normal liver and thyroid function tests, normal creatine kinase levels, but her creatinine levels were mildly elevated. More optimal blood pressure control was recommended for implementation by her referring physician. Her spouse was sampled and found to have normal lipids. The proband’s kindred is shown in the Figure 1. The proband’s father (I-3) was of Irish origin. He had a myocardial infarction at the age of 50 years and died at the age of 54 years of CHD. One of his brothers (I-2) died at the age of 75 years of CHD. The proband’s mother (I-4) had a history of smoking and hypertension, and died at the age of 73 years of lung cancer. No lipid levels were available for these subjects. The proband’s sister (II-4) also had a history of low HDL-C (4–10 mg/dL) and high TGs (200–450 mg/dL), as did one of her daughters (III-5; HDL-C, 4–7 mg/dL and TG, 650–1150 mg/dL). The proband’s sister had a history of obesity and diabetes. However, neither of these subjects had any evidence of heart disease and did not want to be sampled for this study. One brother aged 74 years, had normal lipids, and was in good health.
392 Table 1
Journal of Clinical Lipidology, Vol 9, No 3, June 2015 Lipid, lipoprotein, and apolipoprotein values in cases and controls
Parameter
Proband (II-6)
Age (y) 67 TC (mg/dL) 176 Triglycerides (mg/dL) 215 LDL-C (mg/dL) 122 HDL-C (mg/dL) 8 ApoA-I (mg/dL) 74.7 ApoB (mg/dL) 87 Lp(a) (mg/dL) 11.4 Apo A-I concentrations in HDL subpopulations a-1 (very large) 4.1 a-2 (large) 29.6 a-3 (medium) 13.1 a-4 (small) 16.2 Preb-1 (very small) 6.3
Offspring 1 (III-7)
Offspring 2 (III-8)
Mean 6 SD (%) affected (n 5 3)
Mean 6 SD controls (n 5 20)
39 137 220 95 3 35.8 94 12.0 (mg/dL) 2.9 15.6 4.2 4.9 3.3
35 146 160 100 7 66.8 83 14.15
47.0 6 17.4 153.0 6 20.4 198.0 6 33.3* 106.0 6 14.4 6.0 6 2.6* 59.1 6 20.6* 88.0 6 5.6 12.5 6 3.2
(83.6) (185.0) (101.9) (10.0) (35.0) (107.3) (54.5)
58.0 183.2 107.4 103.6 60.1 169.3 81.7 23.1
6 6 6 6 6 6 6 6
15.1 37.4 37.2 33.0 15.7 32.5 22.6 27.3
3.4 6 0.6* 22.5 6 7.0* 10.1 6 5.1 12.9 6 7.0 4.9 6 1.5
(15.7) (53.6) (76.5) (119.4) (92.5)
21.6 42.0 13.2 10.8 5.3
6 6 6 6 6
4.7 3.5 2.6 2.5 1.7
3.4 22.4 13.1 17.8 5.1
Apo, apolipoprotein; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Lp(a), lipoprotein(a); SD, standard deviation; TC, total cholesterol. Values in parentheses indicate the percentage of the mean normal value based on 10 normal male and 10 normal female controls. *P , .05 as compared with controls.
The proband had 2 sons (III-7 and III-8), both of whom had a history of low HDL-C (less than 20 mg/dL), with no history of CHD or smoking. Subject III-7 was sampled while on atorvastatin 20 mg/d (placed on this medication because of prior significant hyperlipidemia with total serum cholesterol and TG values that were both .250 mg/dL). He also had significant obesity with a body mass index of 41.2 kg/m2. Subject III-8 was sampled off medication and had a body mass index of 25.6 kg/m2. Both these subjects had normal liver, kidney, and thyroid function studies. There were 10 normal male and 10 normal female control subjects used for this study. All subjects were sampled after an overnight fast and were not on medications known to affect lipid metabolism. All females were premenopausal. Their mean age and body mass index was 34.5 years and 25.1 kg/m2, respectively.
Laboratory analysis Lipid, apolipoprotein, and HDL particle profiles Laboratory results on blood obtained after an overnight fast were analyzed at Boston Heart Diagnostics, Framingham, Massachusetts, as previously described.3,4,11 The concentrations of lipids, lipoproteins, apolipoproteins, and HDL particle profiles in the family members and control subjects are shown in Table 1. Compared with controls, the proband and her affected sons had significant increases in serum TG levels that were 185% of control values (P , .05), and significant reductions in serum HDL-C and apoA-I concentrations that were 10% and 35% of control values, respectively (both P , .05). Statistical analysis was carried out using paired t test analysis comparing values in the 3 affected subjects with the 20 control subjects.
HDL particles were analyzed by 2-dimensional gel electrophoresis followed by apoA-I antibody immunoblotting. The concentration of apoA-I in mg/dL within each HDL subpopulation was calculated, as described previously.1,3,4,11,12 These results showed that the mean apoA-I concentrations in very large a-1, large a-2, medium a-3, small a-4, and very small preb-1 HDL particles in affected family members were 15.7%, 53.6%, 76.5%, 119.4%, and 92.5%, respectively, of values in controls. The reductions in the very large a-1 and large a-2 HDL particles were significant (P , .05) compared with controls. In contrast, the unaffected spouse of the proband had normal values for all HDL particle parameters. The percentage of total cholesterol in the esterified form and phospholipid levels were assessed in whole plasma, the non-HDL fraction, and the HDL fraction in affected subjects and controls as previously described in our laboratory at Tufts University.12–15 The mean percentages of total cholesterol in the esterified form in affected subjects in plasma, non-HDL, and HDL were 83%, 91%, and 52% of control values, respectively (only the latter value in HDL was significantly decreased, P , .05). CETP mass levels were measured as previously described in whole plasma, non-HDL, and HDL of affected subjects and controls, and values in affected subjects were 89%, 110%, and 49% of control values, respectively (only the latter value in HDL was significantly decreased, P , .05).15,16 The level of CETP and LCAT activity were measured using plasma and HDL as previously described and were found to be 92% and 71% of controls values (the latter being significant at P , .05).16 The mean levels of phospholipids in plasma, non-HDL, and HDL in affected subjects measured as previously described were 110%, 91%, and 15% of controls values, respectively (only the latter value
Anthanont et al Table 2
ApoA-I variant, cholesterol efflux, and LCAT activity
393
Cholesterol efflux capacity and LCAT activity in the proband’s family
Subject Proband (II-6) III-7 III-8 Mean 6 SD Controls (n 5 20)
Global cholesterol efflux (% per 4 h) (1cAMP) 5.96 4.25 5.76 5.32 6 0.93* (41.4%) 12.86 6 2.87
Cholesterol efflux mediated by (% per 4 h) ABCA1
SR-BI
Ex vivo LCAT activity (% esterified per 4 h)
2.58 2.21 2.30 2.36 6 0.19* (36.5%) 6.46 6 2.19
1.59 0.96 1.86 1.47 6 0.46* (41.0%) 3.59 6 0.86
12.68 12.08 11.72 12.16 6 0.48* (73.4%) 16.57 6 2.19
ABCA1, ATP-binding cassette transporter A1; Apo, apolipoprotein; cAMP, cyclic adenosine monophosphate; 1cAMP, cAMP-treated J774 cells; LCAT, lecithin-cholesterol acyltransferase; SD, standard deviation; SR-BI, scavenger receptor B1. Values in parentheses indicate percentage of values obtained using apoB-depleted control human serum from control subjects. *P , .05 as compared with controls.
in HDL was significantly decreased, P , .05).15 Plasma apoA-II levels were measured by immunoassay as previously described, and mean values in the affected subjects were significantly reduced (P , .05) at 45% of values in controls.15 DNA sequencing Because of our laboratory findings, DNA was isolated from white blood cells obtained from the proband, her 2 sons, and her spouse. The APOA1 gene was sequenced using Sanger sequencing methodology using our core sequencing facility. In this study, the protein sequence numbers refer to the mature apoA-I (243 amino acids), and the nucleotide numbers refer to the reference sequence NM_000039.1 (nucleotide 1 is the A of the ATG-translation initiation codon). DNA sequencing revealed that the proband (II-6) and her 2 sons (III-7 and III-8) were found to be heterozygous for a C to A substitution at nucleotide 517 (c.517C.A). This mutation creates an amino acid substitution changing arginine to serine at residue 149 (p.Arg149Ser). The proband’s spouse (II-7) had a normal APOA1 sequence. In addition, the LCAT gene was sequenced in the proband and was found to be normal. Assessment of cellular cholesterol efflux capacity and ex vivo LCAT activity Cholesterol efflux capacities of serum apoB-depleted samples (3 affected subjects, 1 nonaffected subject, and 20 control subjects) were determined, as described previously, under contract in the Vascular Strategies Laboratory, Plymouth Meeting, Pennsylvania.17 Global and ABCA1mediated cholesterol efflux were measured using mouse J774 macrophages in the presence and absence of cyclic adenosine monophosphate (cAMP). Scavenger receptor B type I (SR-BI)–mediated efflux was measured using rat Fu5AH hepatoma cells. For all assays, cells were labeled by incubation with [3H]-cholesterol and the acetyl-coenzyme A aceyltransferase inhibitor Sandoz 58-035 for 24 hours. Cells were then incubated overnight in 0.2% bovine serum albumin. Results were obtained as previously described.17,18 Ex vivo LCAT activity, the proportion of [3H]-cholesterol
released from cAMP-treated J774 cells to serum HDL during the 4-hour period that was esterified by serum LCAT in the extracellular medium was measured. The cholesterol efflux capacity and ex vivo LCAT activity of affected subjects and controls are shown in Table 2. The mean global cholesterol efflux, ABCA1mediated efflux, and SR-B1–mediated efflux values of affected family members were 53.0%, 46.2%, and 52.1%, respectively, of control values (all significantly reduced, P , .05). In addition, affected family member had mean serum LCAT activity that was 73.4% of control values (also significantly reduced, P , .05).
Discussion The proband in this kindred had severe HDL deficiency, mild hypertriglyceridemia, and detectable large a-1 and a-2 HDL particles by 2-dimensional gel electrophoresis, although the levels of these particles were significantly reduced. The most likely possibility therefore was an apoAI variant because patients with homozygous Tangier disease due to ABCA1 mutations generally have only preb-1 HDL present in plasma, whereas those with homozygous LCAT deficiency generally have only have preb-1 and a-4 HDL present in plasma.1 DNA sequencing revealed a heterozygous missense mutation of APOA1 (c.517C.A, p.Arg149Ser), which we have designated as apoA-IBoston. The proband and her 2 sons had severe HDL deficiency, and all were heterozygous for apoA-IBoston. In addition, the proband’s LCAT gene was found to be normal. The heterozygous form of this mutation has resulted in a dominant negative phenotype because the apoA-I levels in the affected subjects were ,50% of control values. A similar situation has been observed with 7 other apoA-I variants: apoA-IFin, apoA-IZavalla, apoA-IMallorca, apoAISeattle, and apoA-IMilano.2,5–10,19 ApoA-IFin and apoAIZavalla are missense mutations at residue 159 in helix 6, whereas apoA-ISeattle and apoA-IMallorca are deletion mutations within helix 6 and 7, respectively. ApoA-IMilano is characterized by the substitution of arginine by cysteine
394 at residue 173, resulting in the formation of a disulfidelinked apoA-IMilano homodimer, as well as a heterodimer with apoA-II. The proposed mechanism of this group implies that the oligomeric (dimer or tetrameric) apoA-I, constituted both by wild type and mutant, would have a reduced capacity to activate the enzyme LCAT. Moreover, the lack of efficient cholesterol esterification causes the formation of lipid-poor HDL that is rapidly catabolized. ApoA-I plays a role in LCAT activation. The function of LCAT is to transfer a fatty acid from lecithin or phosphatidylcholine to free cholesterol on the surface of lipoproteins to form cholesteryl ester, which migrate into the core of HDL, resulting in the formation of mature, spherical medium and large a migrating HDL particles. It has been established by Sorci-Thomas and Thomas5 as well as other investigators that the central a-helical region (residues 121–186) of apoA-I are involved in LCAT activation.2 It has been reported that 3 arginine residues in this region (Arg-149, Arg-153, and Arg-160) are critical for the ability of apoA-I to activate LCAT.18 In this kindred, affected family members had mean apoA-II, apoA-I, HDL phospholipid, and HDL-C levels that were all significantly reduced at 45%, 35%, 15%, and 10%, respectively of control values. Moreover the percentage of HDL-C as cholesteryl ester and CETP mass in HDL were both significantly reduced to 52% and 49% of control values, and the levels of apoA-I in very large a-1 and large a-2 HDL particles were also significantly reduced at 16% and 54% of control values, respectively. All affected subjects in the kindred were found to be heterozygous for an apoA-I mutation causing the replacement of an arginine with a serine at residue 149 (Arg149Ser). Based on prior studies, it would be predicted that this amino acid substitution would have an effect on the ability of apoA-I to activate LCAT. HDL functionality studies documented that the mean global cholesterol efflux, ABCA1-mediated efflux, and SR-B1–mediated efflux values of affected family members were significantly reduced to 53%, 46%, and 52%, respectively, of control values. These decreases are commensurate with decreases in HDL particle mass. Moreover, mean ex vivo apoB-depleted serum LCAT activity was significantly reduced at 73.4% of control values. This assay reflects the proportion of labeled cholesterol released from cAMP-treated J774 cells to serum HDL during a 4-hour period that was esterified by serum LCAT. The overall finding in this kindred with no evidence of premature CHD in the proband or her affected younger sons are consistent with observations in 3 other kindreds with heterozygous apoA-I missense mutations (Leu141Arg)Pisa, (Arg160Leu)Oslo, and (Pro165Arg), which were associated with low HDL-C, decreased LCAT activity, decreased cellular cholesterol efflux, and no evidence of premature CHD. It should also be noted that neither we nor others have observed premature CHD in patients with familial LCAT deficiency despite very low HDL-C levels.12,14
Journal of Clinical Lipidology, Vol 9, No 3, June 2015 However, the proband’s father did die of premature CHD at the age of 54 years. An important function of HDL particles is the promotion of global cellular cholesterol efflux, which has been associated with CHD risk.20,21 Cellular cholesterol efflux can be assessed in a global fashion, as well as by also examining ABCA1-mediated and SR-BI–mediated efflux, as was done in this kindred. The primary HDL particle serving as an acceptor of cholesterol and phospholipids via the ABCA1 transporter is very small lipid-poor preb-1 HDL.18 In contrast, the preferred HDL particle for bidirectional cholesterol flux mediated via the SR-BI transporter is very large lipid-rich a-1 HDL.18 In our view, the SR-BI–mediated cholesterol efflux in this kindred was decreased because of a significant decrease in very large a-1 HDL particles. We postulate that the reduction in the ABCA1-mediated cholesterol efflux in this kindred was because of both a modest decrease in preb-1 HDL and a reduction in LCAT activity and cholesterol esterification. To our knowledge, there is no prior report of this mutation in humans.
Financial disclosures The authors have no conflicts of interest to disclose. P.A. was a postdoctoral research fellow from Thammasat University, Thailand. B.F.A., E.P., and E.J.S. are employees and have ownership interest in Boston Heart Diagnostics, Framingham, Massachusetts. This research was supported by the US Department of Agriculture Research Service Contract 53-3K-06 (E.J.S.) and Project Grant P50 HL083813-01 from the National Institutes of Health (E.J.S.).
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ApoA-I variant, cholesterol efflux, and LCAT activity
8. Daum U, Leren TP, Langer C, et al. Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I (P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers. J Lipid Res. 1999;40:486–494. 9. Miccoli R, Zhu Y, Daum U, et al. A natural apolipoprotein A-I variant, apoA-I (L141R)Pisa, interferes with the formation of alpha-high density lipoproteins (HDL), but not with the formation of pre beta 1-HDL and influences efflux of cholesterol into plasma. J Lipid Res. 1997;38:1242–1253. 10. Miccoli R, Bertolotto A, Navalesi R, et al. Compound heterozygosity for a structural apolipoprotein A-I variant, apo A-I (L141R) Pisa, and an apolipoprotein A-I null allele in patients with absence of HDL cholesterol, corneal opacifications, and coronary heart disease. Circulation. 1996;94:1622–1628. 11. Asztalos BF, Cupples LA, Demissie S, et al. High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants in the Framingham Offspring Study. Arterioscler Thromb Vasc Biol. 2004;24:2181–2187. 12. Roshan B, Ganda OP, Desilva R, et al. Homozygous lecithin:cholesterol acyltransferase (LCAT) deficiency due to a new loss of function mutation and review of the literature. J Clin Lipidol. 2011;5: 493–499. 13. Dimick SM, Sallee B, Asztalos BF, et al. A kindred with fish eye disease, corneal opacities, marked high-density lipoprotein deficiency, and statin therapy. J Clin Lipidol. 2014;8:223–230.
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