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A 60-y-old chylomicronemia patient homozygous for missense mutation (G188E) in the lipoprotein lipase gene showed no accelerated atherosclerosis
Clinica Chimica Acta 386 (2007) 100 – 104 www.elsevier.com/locate/clinchim
A 60-y-old chylomicronemia patient homozygous for missense mutation (G188E) in the lipoprotein lipase gene showed no accelerated atherosclerosis Tetsu Ebara a,b , Yoriko Endo a , Shouichi Yoshiike b , Masatomi Tsuji b , Susumu Taguchi b , Toshio Murase a , Minoru Okubo a,c,⁎ b
a Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan Department of Internal Medicine, Showa University Northern Yokohama hospital, 35-1 Chigasaki, Tsuzuki-ku, Yokohama, Kanagawa 224-8503, Japan c Department of Endocrinology and Metabolism, Toranomon Hospital, Tokyo, Japan
Received 10 May 2007; received in revised form 25 August 2007; accepted 27 August 2007 Available online 1 September 2007
Abstract Background: Familial lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder caused by mutations in the LPL gene. Patients with LPL deficiency have chylomicronemia; however, whether they develop accelerated atherosclerosis remains unclear. Methods: We investigated clinical and mutational characteristics of a 60-y-old Japanese patient with chylomicronemia. Results: The patient's fasting plasma triglyceride levels were N 9.0 mmol/l. In postheparin plasma, one fifth of the normal LPL protein mass was present; however, LPL activity was undetectable. Molecular analysis of the LPL gene showed the patient to be a homozygote of missense mutation replacing glycine with glutamine at codon 188 (G188E), which had been known to produce mutant LPL protein lacking lipolytic activity. Ultrasonographic examination of the patient's carotid and femoral arteries showed no accelerated atherosclerosis. Moreover, 64-slice mechanical multidetector-row computer tomography (MDCT) angiography did not detect any accelerated atherosclerotic lesions in the patient's coronary arteries. The patient had none of the risk factors such as smoking, hypertension, and diabetes. Conclusions: Our case suggests that accelerated atherosclerosis may not develop in patients with LPL deficiency, when they have no risk factors. © 2007 Elsevier B.V. All rights reserved. Keywords: Lipoprotein lipase deficiency; Chylomicronemia; Point mutation; Atherosclerosis; Mechanical multidetector-row computer tomography
1. Introduction Lipoprotein lipase (LPL) is the primary enzyme responsible for the catabolism of triglyceride-rich lipoprotein particles. The enzyme hydrolyses triglycerides in chylomicrons and VLDL using apolipoprotein (apo) CII as a cofactor [1]. LPL is mainly synthesized by adipose tissue and muscle, transported, and anchored to the luminal surface of the capillary endothelium by heparin sulfate proteoglycans. It is postulated that LPL functions as a potent bridge between lipoproteins and proteoglycans on vessel walls, retaining atherogenic lipoproteins on endothelial cells, exclusive of its enzymatic activity [2]. Some animal model ⁎ Corresponding author. Okinaka Memorial Institute for Medical Research, 22-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111; fax: +81 3 3582 7068. E-mail address: [email protected] (M. Okubo). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.08.011
experiments, using mice and rabbits, showed that macrophage LPL promotes foam cell formation and atherosclerosis [3,4]. On the contrary, systemic overexpression of LPL cDNA in mice is protective against atherosclerosis [5]. There are species differences in susceptibility to atherosclerosis, and whether LPL protein is pro-atherogenic or anti-atherogenic remains unclear in humans. Defects in LPL-apo CII lipolysis system result in severe accumulation of chylomicrons in plasma. Familial LPL deficiency is a rare autosomal recessive disorder, estimated to be about one in one million persons. Patients with LPL deficiency show severe hypertriglyceridemia, eruptive xanthoma, hepatosplenomegaly, and recurrent attacks of pancreatitis. Many patients died from pancreatitis at an early age in the past, but today, LPL deficiency is diagnosed in childhood and the maintenance of a low-fat diet enables patients lead a longer life [1]. Whether patients with LPL deficiency develop premature atherosclerosis is, consequently, becoming a concern.
T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
101
Table 1 Biochemical data for a lipoprotein lipase deficient patient Variable Age (y) Triglyceride (mmol/l) Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Apo AI (mg/dl) Apo B (mg/dl) Apo CII (mg/dl) Apo CIII (mg/dl) Apo E (mg/dl) Apo E genotype LPL mass (ng/ml) HbA1c (%)
Admission
Day 10
18 days after discharge
10 months after discharge
Normal range
0.3
60 34.5 6.9 1.6 0.1
60 9.7 ND ND ND 62 96 5.3 13.3 8.2 3/3 45
60 9.0 4.4 0.8 0.5
60 5.8 3.2 0.4 0.5 81 67 7.6 12.9 8.7
b1.7 b5.7 b3.6 0.9–1.8 89–192 40–128 1.2–6.4 3.4–12.6 2.2–6.9
5.0
4.8
49 17.1 4.0
51 27.3 5.7
58 27.1 5.2
1.1
0.2
223 ± 66 a b5.8
ND, not determined. a Normal values are expressed as mean ± S.D.
It has generally been thought that patients with LPL deficiency do not predispose to develop atherosclerosis, since chylomicrons were believed to be too large to penetrate vessel walls. A study using a mouse model for chylomicronemia supports this view [6]. However, Benlian et al. raised a question on this assumption, based on an observation that premature atherosclerosis develops in four patients with LPL deficiency, and suggested that LPL deficiency may increase susceptibility to atherosclerosis [7]. In contrast, we reported that there was no evidence of accelerated atherosclerosis in a 66-y-old chylomicronemia patient homozygous for nonsense mutation [8]. It has thus been suggested a hypothesis that type of LPL mutation may affect atherosclerosis: LPL mutations leading to null LPL protein do not accelerate atherosclerosis, while missense mutations with retained LPL protein promote atherosclerosis [7–9]. Here we report a 60-y-old patient with LPL deficiency, who had non-catalytic LPL protein and did not have accelerated atherosclerosis. Our case provides a unique opportunity to explore clinical atherosclerosis associated with homozygous LPL deficiency. 2. Materials and methods 2.1. Patient A 60-y-old Japanese female was assessed in this study. She was brought up in the Kanto district in Japan and received a medical checkup for the first time when she was 49 y. Her plasma triglyceride concentration was then 17.1 mmol/l. Hypertriglyceridemia was, in retrospect, evident in her medical record as shown in Table 1, but she did not pay attention to it. Her body mass index was 22.4 kg/m2 and her blood pressure was within normal range. The patient's fasting blood glucose and hemoglobin A1c levels were also normal. She has never experienced angina pectoris. At the age of 60 y, she suffered from acute pancreatitis. Before the admission, she attended several parties and took large amounts of meals containing fat and carbohydrates, which she used to refrain from. On dietary habit, she has taken a traditional Japanese diet, which consisted of 17% protein, 23% fat, and 60% carbohydrate, that is, equivalent of diet recommended by the national cholesterol education program [10]. After discharge, she is keeping on low-fat diet b10% of total calories. She has no habit of smoking and drinking alcohol. Her parents were first cousins. However, none of her relatives had been diagnosed with coronary heart disease or pancreatitis, to our knowledge.
2.2. Lipids and apoproteins A blood sample was obtained from the patient after an overnight fast. Plasma total cholesterol and triglyceride concentrations were measured by enzymatic methods. High-density lipoprotein (HDL) cholesterol was quantified in the plasma after the polyanion precipitation of apo B-containing lipoproteins. Low-density lipoprotein (LDL) cholesterol was measured by a homogeneous method using a direct LDL-cholesterol assay kit (Daiichi Pure Chemicals Co., Tokyo, Japan) [11]. Plasma apo AI, AII, B, CII, CIII, and E were determined by an immunoturbidometric assay (Daiichi Pure Chemicals Co., Tokyo, Japan), according to the manufacturer's instructions.
2.3. LPL mass and activity Postheparin plasma from the patient was collected 10 min after a bolus injection of heparin (30 units/kg body weight). The LPL mass was determined using a sandwich ELISA (Dai-Nippon Pharmaceutical, Osaka, Japan), according to the manufacturer's instructions. LPL activity and hepatic triglyceride lipase (HL) activity were assayed as described previously [12].
2.4. Nucleotide sequence analysis of the LPL gene Genomic DNA was extracted from the peripheral blood sample after informed consent was obtained from the patient. The PCR fragments containing each exon and exon–intron boundary of the LPL gene were amplified as described previously [13]. Direct sequencing of the PCR products was performed using a Big Dye Terminator cycle sequencing kit on a genetic analyzer PRISM 310 (Applied Biosystems, CA, USA). The nucleotides of LPL cDNA were numbered from the AUG codon according to GenBank accession no. NM_000237. The study was approved by our ethics committee.
2.5. Detection of the mutation by restriction fragment length polymorphism (RFLP) To verify the G-to-A transition at nucleotide 818 in exon 5, a pair of a sense primer (5′-atc tgt gtt cct gct ttt ttc c-3′) in intron 4 and an antisense primer (5′aag agt cac att taa ttc gct tc-3′) in intron 5 were used. PCR was carried out by 30 cycles of denaturation, annealing, and extension at 94 °C for 30 s, 54 °C for 1 min, and 72 °C for 2 min, respectively. The fragments then were digested with restriction endonuclease Ava II and analyzed on a 5% polyacrylamide gel. In the presence of the mutation, the 256-bp PCR product was uncleaved by Ava II, whereas it was cleaved into 168- and 88-bp fragments with the normal sequence.
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T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
Table 2 Reports focusing on atherosclerosis of LPL deficient patients Patient no. Age Sex LPL mutation LPL mass a Cervical atherosclerosis Coronary atherosclerosis Smoking Hypertension Glucose intolerance Reporter 1 2
75 52
M M
3
54
F
4
67
M
5
62
F
6 7
66 55
F M
8
53
M
9
60
F
n.d. G188R/ G188R G188E/ R243C Frameshift/ L286P T101A/ D250N Y61X/Y61X L303F/ L303F delG916/ delG916 G188E/ G188E
n.d. 73
n.d. +
+ +
n.d. +
n.d. −
+ −
Hoeg Benlian
45.2
+
+
+
−
−
Benlian
33
+
n.d.
+
+
+
Benlian
26.6
+
+
−
−
+
Benlian
0 24.4
− −
− +
− +
− +
− −
Ebara Saika
0
−
−
+
−
+
Kawashiri
20
−
−
−
−
−
This study
n.d., not described. a Percentage of the mean normal value.
2.6. Apo E genotype Apo E genotype was determined by digestion of PCR-amplified fragments with restriction enzyme Hha I as described [14].
2.7. Clinical examination of the patient The exercise-tolerance electrocardiogram was judged according to the Minnesota standards. Intima–media thickness (IMT) was measured at the patient's bilateral common and internal carotid arteries, and femoral arteries by standard B-mode ultrasonography as described previously [8]. In order to screen coronary artery lesions, 64-slice mechanical multidetector-row computer tomography (MDCT) angiography (Toshiba, Tokyo, Japan) was performed for the patient. A volume data set was acquired covering the region from the pulmonary hilum to the diaphragmatic surface of the heart. Computed tomography gantry rotation time was 330 ms. Tube voltage and effective tube current–time product were set to 120 kV and 880 mA s.
3. Results Table 1 shows the biochemical data for the patient. The plasma triglyceride concentration was significantly high, and the HDL-cholesterol level was below normal range. Chylomicrons were observed in the patient's plasma after the overnight storage at 4 °C. The patient's plasma apo CII level was not deficient, and apo E genotype was E3/E3. The patient's HbA1c level was normal. In postheparin plasma, the patient's LPL mass was 45 ng/ml, i.e., one fifth of the mean normal value. However, her LPL activity was undetectable (normal: 6.4 ± 2.1 μmol FFA/ml/ h), whereas her HL activity was within normal range (6.6 μmol FFA/ml/h; normal: 8.8 ± 2.9). The direct sequencing analysis showed a G-to-A substitution at nucleotide 818, the second base of codon 188, in exon 5 of the patient's LPL gene. This point mutation replaced glycine (GGG) with glutamic acid (GAG) (G188E). In Ava II RFLP analysis, the patient had a 256-bp fragment alone, demonstrating that the patient was homozygous for the G188E mutation. Homozygosity was compatible with the fact that her parents
were first cousins. These findings allowed us to diagnose the patient as having with LPL deficiency. The ultrasonogram of the carotid arteries indicated that this patient did not exhibit accelerated atherosclerosis. Neither stenosis nor calcification was detected. Ultrasonographic examination of femoral arteries did not detect accelerated atherosclerosis either. The electrocardiogram after exercise-tolerance testing was normal. MDCT angiography for this patient showed a thin atheromatous plaque in the right coronary artery. However, this lesion was consistent with her age and no significant stenosis was detected in any other segments of coronary arteries. These findings indicated that the patient had no clinical signs of accelerated atherosclerosis. 4. Discussion We showed the patient to be homozygous for missense mutation (G188E) in the LPL gene, and diagnosed her with LPL deficiency. Previous reports on patients with G188E have demonstrated that the mutation causes LPL deficiency. In Caucasian, G188E has been shown as one of the prevalent LPL mutations [15,16]. Furthermore, G188E has been detected in LPL deficient patients of non-Caucasian descent. Our patient is the third case with G188E and the eldest in Japan, since 2 patients under 1 y with the same mutation have been reported [17,18]. Patients homozygous for G188E manifested reduced amount of LPL protein without LPL activity [19]. Moreover, in vitro expression studies established that G188E produced mutant LPL protein completely lacking lipolytic activity [20–22]. Our patient had one fifth of the normal LPL protein mass and no LPL activity. These findings were compatible with LPL deficiency with G188E mutation. In complete LPL deficient patients, reports focusing on whether they have accelerated atherosclerosis have been limited (Table 2) [7,8,23–25]. Although chylomicronemia is not believed to be associated with atherosclerosis, this hypothesis has been called into question by Benlian et al. They observed premature atherosclerosis in 4 patients (patients 2–5 in Table 2) and
T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
proposed that defective lipolysis may increase susceptibility to atherosclerosis in humans [7]. Saika et al. observed that a patient with missense mutation L303F suffered from coronary artery disease and severe systemic atherosclerosis (patient 7) [24]. On the other hand, we described a patient homozygous for nonsense mutation who had no evidence of accelerated atherosclerosis (patient 6) [8]. In addition, Kawashiri et al. showed that a patient with another nonsense mutation had no clinically significant atherosclerotic lesions (patient 8) [25]. Accordingly, it has been argued that atherosclerosis among those patients is attributable to the type of LPL mutations [7–9,24,25]. Missense mutation resulting in the presence of LPL protein (even though it has no catalytic activity) develops accelerated atherosclerosis, while nonsense mutations do not accelerate atherosclerosis because of the lack of LPL protein. LPL deficient patient with no mass (patients 6 and 8) had no evidence of accelerated atherosclerosis, and those with LPL mass (patients 2–5, 7) had accelerated atherosclerosis. In contrast to the hypothesis that catalytically inactive LPL protein is pro-atherogenic, our patient homozygous for missense mutation G188E did not have accelerated atherosclerosis. Carotid, femoral, and coronary arteries in the patient were assessed by using ultrasonography and 64-slice MDCT angiography, and no evidence of accelerated atherosclerosis was found. This case suggests that the type of LPL mutation may not be a major factor determining progression of atherosclerosis in LPL deficient patients. Presumably, classic risk factors, such as smoking, hypertension, and glucose intolerance, play a larger role on atherosclerosis than LPL. As far as risk factors are concerned, patients 2–4 with missense mutations had some of risk factors. Patient 3 was a compound heterozygote of G188E and R243C but a smoker, who had angina pectoris. However, patient 8 did not show accelerated atherosclerosis, despite risk factors. Further study is necessary to clarify the impact of LPL mutation types on atherosclerosis in LPL deficient patients. Decreased LDL-cholesterol levels may prevent premature atherosclerosis in LPL deficient patient. LPL plays a key role in production of LDL and the defect of LPL function leads to the impairment of the conversion from VLDL to LDL. Zambon et al. reported a patient who is both heterozygous for FH and homozygous for LPL deficiency and described that the LDL-cholesterol level was significantly lower than the levels of noromolipidemic relatives [26]. The absence of LPL activity lowers plasma LDLcholesterol levels and the susceptibility to atherosclerosis. In summary, the molecular basis of a 60-y-old patient with chylomicronemia has been characterized, and our case suggests that accelerated atherosclerosis may not develop in patients with LPL deficiency, when they have no risk factors.
[10]
Acknowledgement
[19]
This study was supported in part by a Research Grant from the Takeda Science Foundation.
[2] [3]
[4]
[5]
[6]
[7]
[8]
[9]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[20]
References [21] [1] Brunzell J, Deeb S. Familial lipoprotein lipase deficiency, apo C-II deficiency, and hepatic lipase deficiency. In: Scriver C, Beaudet A, Sly W,
103
Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. p. 2789–816. Merkel M, Eckel RH, Goldberg IJ. Lipoprotein lipase: genetics, lipid uptake, and regulation. J Lipid Res 2002;43:1997–2006. Babaev VR, Fazio S, Gleaves LA, Carter KJ, Semenkovich CF, Linton MF. Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo. J Clin Invest 1999;103:1697–705. Wu X, Wang J, Fan J, et al. Localized vessel expression of lipoprotein lipase in rabbits leads to rapid lipid deposition in the balloon-injured arterial wall. Atherosclerosis 2006;187:65–73. Yagyu H, Ishibashi S, Chen Z, et al. Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice. J Lipid Res 1999;40:1677–85. Ebara T, Ramakrishnan R, Steiner G, Shachter NS. Chylomicronemia due to apolipoprotein CIII overexpression in apolipoprotein E-null mice. Apolipoprotein CIII-induced hypertriglyceridemia is not mediated by effects on apolipoprotein E. J Clin Invest 1997;99:2672–81. Benlian P, De Gennes JL, Foubert L, Zhang H, Gagne SE, Hayden M. Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N Engl J Med 1996;335:848–54. Ebara T, Okubo M, Horinishi A, Adachi M, Murase T, Hirano T. No evidence of accelerated atherosclerosis in a 66-yr-old chylomicronemia patient homozygous for the nonsense mutation (Tyr61→stop) in the lipoprotein lipase gene. Atherosclerosis 2001;159:375–9. Foubert L, De Gennes JL, Benlian P, Truffert J, Miao L, Hayden MR. Compound heterozygosity for frameshift mutations in the gene for lipoprotein lipase in a patient with early-onset chylomicronemia. Hum Mutat 1998;Suppl 1:S141–4. Third report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report. Circulation 2002;106:3143–421. Sakaue T, Hirano T, Yoshino G, Sakai K, Takeuchi H, Adachi M. Reactions of direct LDL-cholesterol assays with pure LDL fraction and IDL: comparison of three homogeneous methods. Clin Chim Acta 2000;295:97–106. Murase T, Yamada N, Ohsawa N, Kosaka K, Morita S, Yoshida S. Decline of postheparin plasma lipoprotein lipase in acromegalic patients. Metabolism 1980;29:666–72. Okubo M, Inoue S, Horinishi A, et al. Detection of a new compound heterozygote (del G916/G1401A) for lipoprotein lipase deficiency and a comparative haplotype analysis of the mutant lipoprotein lipase gene from Japanese patients. Atherosclerosis 1999;144:443–7. Hixon JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hha I. J Lipid Res 1990;31:545–8. Bergeron J, Normand T, Bharucha A, et al. Prevalence, geographical distribution and genealogical investigations of mutation 188 of lipoprotein lipase gene in the French Canadian population of Quebec. Clin Genet 1992;41:206–10. Gilbert B, Rouis M, Griglio S, de Lumley L, Laplaud P. Lipoprotein lipase (LPL) deficiency: a new patient homozygote for the preponderant mutation Gly188Glu in the human LPL gene and review of reported mutations: 75% are clustered in exons 5 and 6. Ann Genet 2001;44:25–32. Takagi A, Ikeda Y, Tachi K, Shinozuka T, Yamamoto A. Identification of compound heterozygous mutations (G188E/W382X) of lipoprotein lipase gene in a Japanese infant with hyperchylomicronemia: the G188E mutation was newly identified in Japanese. Clin Chim Acta 1999;285:143–54. Yoshida T, Gotoda T, Okubo M, et al. A Japanese patient with lipoprotein lipase deficiency homozygous for the Gly188Glu mutation prevalent worldwide. J Atheroscler Thromb 2000;7:45–9. Monsalve MV, Henderson H, Roederer G, et al. A missense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries. J Clin Invest 1990;86:728–34. Emi M, Wilson DE, Iverius PH, et al. Missense mutation (Gly–Glu188) of human lipoprotein lipase imparting functional deficiency. J Biol Chem 1990;265:5910–6. Hata A, Ridinger DN, Sutherland SD, et al. Missense mutations in exon 5 of the human lipoprotein lipase gene. Inactivation correlates with loss of dimerization. J Biol Chem 1992;267:20132–9.
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[22] Peterson J, Ayyobi AF, Ma Y, et al. Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene. J Lipid Res 2002;43:398–406. [23] Hoeg JM, Osborne Jr JC, Gregg RE, Brewer Jr HB. Initial diagnosis of lipoprotein lipase deficiency in a 75-year-old man. Am J Med 1983;75:889–92. [24] Saika Y, Sakai N, Takahashi M, et al. Novel LPL mutation (L303F) found in a patient associated with coronary artery disease and severe systemic atherosclerosis. Eur J Clin Invest 2003;33:216–22.
[25] Kawashiri MA, Higashikata T, Mizuno M, et al. Long-term course of lipoprotein lipase (LPL) deficiency due to homozygous LPL(Arita) in a patient with recurrent pancreatitis, retained glucose tolerance, and atherosclerosis. J Clin Endocrinol Metab 2005;90:6541–4. [26] Zambon A, Torres A, Bijvoet S, et al. Prevention of raised low-density lipoprotein cholesterol in a patient with familial hypercholesterolaemia and lipoprotein lipase deficiency. Lancet 1993;341:1119–21.
A 60-y-old chylomicronemia patient homozygous for missense mutation (G188E) in the lipoprotein lipase gene showed no accelerated atherosclerosis Tetsu Ebara a,b , Yoriko Endo a , Shouichi Yoshiike b , Masatomi Tsuji b , Susumu Taguchi b , Toshio Murase a , Minoru Okubo a,c,⁎ b
a Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan Department of Internal Medicine, Showa University Northern Yokohama hospital, 35-1 Chigasaki, Tsuzuki-ku, Yokohama, Kanagawa 224-8503, Japan c Department of Endocrinology and Metabolism, Toranomon Hospital, Tokyo, Japan
Received 10 May 2007; received in revised form 25 August 2007; accepted 27 August 2007 Available online 1 September 2007
Abstract Background: Familial lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder caused by mutations in the LPL gene. Patients with LPL deficiency have chylomicronemia; however, whether they develop accelerated atherosclerosis remains unclear. Methods: We investigated clinical and mutational characteristics of a 60-y-old Japanese patient with chylomicronemia. Results: The patient's fasting plasma triglyceride levels were N 9.0 mmol/l. In postheparin plasma, one fifth of the normal LPL protein mass was present; however, LPL activity was undetectable. Molecular analysis of the LPL gene showed the patient to be a homozygote of missense mutation replacing glycine with glutamine at codon 188 (G188E), which had been known to produce mutant LPL protein lacking lipolytic activity. Ultrasonographic examination of the patient's carotid and femoral arteries showed no accelerated atherosclerosis. Moreover, 64-slice mechanical multidetector-row computer tomography (MDCT) angiography did not detect any accelerated atherosclerotic lesions in the patient's coronary arteries. The patient had none of the risk factors such as smoking, hypertension, and diabetes. Conclusions: Our case suggests that accelerated atherosclerosis may not develop in patients with LPL deficiency, when they have no risk factors. © 2007 Elsevier B.V. All rights reserved. Keywords: Lipoprotein lipase deficiency; Chylomicronemia; Point mutation; Atherosclerosis; Mechanical multidetector-row computer tomography
1. Introduction Lipoprotein lipase (LPL) is the primary enzyme responsible for the catabolism of triglyceride-rich lipoprotein particles. The enzyme hydrolyses triglycerides in chylomicrons and VLDL using apolipoprotein (apo) CII as a cofactor [1]. LPL is mainly synthesized by adipose tissue and muscle, transported, and anchored to the luminal surface of the capillary endothelium by heparin sulfate proteoglycans. It is postulated that LPL functions as a potent bridge between lipoproteins and proteoglycans on vessel walls, retaining atherogenic lipoproteins on endothelial cells, exclusive of its enzymatic activity [2]. Some animal model ⁎ Corresponding author. Okinaka Memorial Institute for Medical Research, 22-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111; fax: +81 3 3582 7068. E-mail address: [email protected] (M. Okubo). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.08.011
experiments, using mice and rabbits, showed that macrophage LPL promotes foam cell formation and atherosclerosis [3,4]. On the contrary, systemic overexpression of LPL cDNA in mice is protective against atherosclerosis [5]. There are species differences in susceptibility to atherosclerosis, and whether LPL protein is pro-atherogenic or anti-atherogenic remains unclear in humans. Defects in LPL-apo CII lipolysis system result in severe accumulation of chylomicrons in plasma. Familial LPL deficiency is a rare autosomal recessive disorder, estimated to be about one in one million persons. Patients with LPL deficiency show severe hypertriglyceridemia, eruptive xanthoma, hepatosplenomegaly, and recurrent attacks of pancreatitis. Many patients died from pancreatitis at an early age in the past, but today, LPL deficiency is diagnosed in childhood and the maintenance of a low-fat diet enables patients lead a longer life [1]. Whether patients with LPL deficiency develop premature atherosclerosis is, consequently, becoming a concern.
T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
101
Table 1 Biochemical data for a lipoprotein lipase deficient patient Variable Age (y) Triglyceride (mmol/l) Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Apo AI (mg/dl) Apo B (mg/dl) Apo CII (mg/dl) Apo CIII (mg/dl) Apo E (mg/dl) Apo E genotype LPL mass (ng/ml) HbA1c (%)
Admission
Day 10
18 days after discharge
10 months after discharge
Normal range
0.3
60 34.5 6.9 1.6 0.1
60 9.7 ND ND ND 62 96 5.3 13.3 8.2 3/3 45
60 9.0 4.4 0.8 0.5
60 5.8 3.2 0.4 0.5 81 67 7.6 12.9 8.7
b1.7 b5.7 b3.6 0.9–1.8 89–192 40–128 1.2–6.4 3.4–12.6 2.2–6.9
5.0
4.8
49 17.1 4.0
51 27.3 5.7
58 27.1 5.2
1.1
0.2
223 ± 66 a b5.8
ND, not determined. a Normal values are expressed as mean ± S.D.
It has generally been thought that patients with LPL deficiency do not predispose to develop atherosclerosis, since chylomicrons were believed to be too large to penetrate vessel walls. A study using a mouse model for chylomicronemia supports this view [6]. However, Benlian et al. raised a question on this assumption, based on an observation that premature atherosclerosis develops in four patients with LPL deficiency, and suggested that LPL deficiency may increase susceptibility to atherosclerosis [7]. In contrast, we reported that there was no evidence of accelerated atherosclerosis in a 66-y-old chylomicronemia patient homozygous for nonsense mutation [8]. It has thus been suggested a hypothesis that type of LPL mutation may affect atherosclerosis: LPL mutations leading to null LPL protein do not accelerate atherosclerosis, while missense mutations with retained LPL protein promote atherosclerosis [7–9]. Here we report a 60-y-old patient with LPL deficiency, who had non-catalytic LPL protein and did not have accelerated atherosclerosis. Our case provides a unique opportunity to explore clinical atherosclerosis associated with homozygous LPL deficiency. 2. Materials and methods 2.1. Patient A 60-y-old Japanese female was assessed in this study. She was brought up in the Kanto district in Japan and received a medical checkup for the first time when she was 49 y. Her plasma triglyceride concentration was then 17.1 mmol/l. Hypertriglyceridemia was, in retrospect, evident in her medical record as shown in Table 1, but she did not pay attention to it. Her body mass index was 22.4 kg/m2 and her blood pressure was within normal range. The patient's fasting blood glucose and hemoglobin A1c levels were also normal. She has never experienced angina pectoris. At the age of 60 y, she suffered from acute pancreatitis. Before the admission, she attended several parties and took large amounts of meals containing fat and carbohydrates, which she used to refrain from. On dietary habit, she has taken a traditional Japanese diet, which consisted of 17% protein, 23% fat, and 60% carbohydrate, that is, equivalent of diet recommended by the national cholesterol education program [10]. After discharge, she is keeping on low-fat diet b10% of total calories. She has no habit of smoking and drinking alcohol. Her parents were first cousins. However, none of her relatives had been diagnosed with coronary heart disease or pancreatitis, to our knowledge.
2.2. Lipids and apoproteins A blood sample was obtained from the patient after an overnight fast. Plasma total cholesterol and triglyceride concentrations were measured by enzymatic methods. High-density lipoprotein (HDL) cholesterol was quantified in the plasma after the polyanion precipitation of apo B-containing lipoproteins. Low-density lipoprotein (LDL) cholesterol was measured by a homogeneous method using a direct LDL-cholesterol assay kit (Daiichi Pure Chemicals Co., Tokyo, Japan) [11]. Plasma apo AI, AII, B, CII, CIII, and E were determined by an immunoturbidometric assay (Daiichi Pure Chemicals Co., Tokyo, Japan), according to the manufacturer's instructions.
2.3. LPL mass and activity Postheparin plasma from the patient was collected 10 min after a bolus injection of heparin (30 units/kg body weight). The LPL mass was determined using a sandwich ELISA (Dai-Nippon Pharmaceutical, Osaka, Japan), according to the manufacturer's instructions. LPL activity and hepatic triglyceride lipase (HL) activity were assayed as described previously [12].
2.4. Nucleotide sequence analysis of the LPL gene Genomic DNA was extracted from the peripheral blood sample after informed consent was obtained from the patient. The PCR fragments containing each exon and exon–intron boundary of the LPL gene were amplified as described previously [13]. Direct sequencing of the PCR products was performed using a Big Dye Terminator cycle sequencing kit on a genetic analyzer PRISM 310 (Applied Biosystems, CA, USA). The nucleotides of LPL cDNA were numbered from the AUG codon according to GenBank accession no. NM_000237. The study was approved by our ethics committee.
2.5. Detection of the mutation by restriction fragment length polymorphism (RFLP) To verify the G-to-A transition at nucleotide 818 in exon 5, a pair of a sense primer (5′-atc tgt gtt cct gct ttt ttc c-3′) in intron 4 and an antisense primer (5′aag agt cac att taa ttc gct tc-3′) in intron 5 were used. PCR was carried out by 30 cycles of denaturation, annealing, and extension at 94 °C for 30 s, 54 °C for 1 min, and 72 °C for 2 min, respectively. The fragments then were digested with restriction endonuclease Ava II and analyzed on a 5% polyacrylamide gel. In the presence of the mutation, the 256-bp PCR product was uncleaved by Ava II, whereas it was cleaved into 168- and 88-bp fragments with the normal sequence.
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T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
Table 2 Reports focusing on atherosclerosis of LPL deficient patients Patient no. Age Sex LPL mutation LPL mass a Cervical atherosclerosis Coronary atherosclerosis Smoking Hypertension Glucose intolerance Reporter 1 2
75 52
M M
3
54
F
4
67
M
5
62
F
6 7
66 55
F M
8
53
M
9
60
F
n.d. G188R/ G188R G188E/ R243C Frameshift/ L286P T101A/ D250N Y61X/Y61X L303F/ L303F delG916/ delG916 G188E/ G188E
n.d. 73
n.d. +
+ +
n.d. +
n.d. −
+ −
Hoeg Benlian
45.2
+
+
+
−
−
Benlian
33
+
n.d.
+
+
+
Benlian
26.6
+
+
−
−
+
Benlian
0 24.4
− −
− +
− +
− +
− −
Ebara Saika
0
−
−
+
−
+
Kawashiri
20
−
−
−
−
−
This study
n.d., not described. a Percentage of the mean normal value.
2.6. Apo E genotype Apo E genotype was determined by digestion of PCR-amplified fragments with restriction enzyme Hha I as described [14].
2.7. Clinical examination of the patient The exercise-tolerance electrocardiogram was judged according to the Minnesota standards. Intima–media thickness (IMT) was measured at the patient's bilateral common and internal carotid arteries, and femoral arteries by standard B-mode ultrasonography as described previously [8]. In order to screen coronary artery lesions, 64-slice mechanical multidetector-row computer tomography (MDCT) angiography (Toshiba, Tokyo, Japan) was performed for the patient. A volume data set was acquired covering the region from the pulmonary hilum to the diaphragmatic surface of the heart. Computed tomography gantry rotation time was 330 ms. Tube voltage and effective tube current–time product were set to 120 kV and 880 mA s.
3. Results Table 1 shows the biochemical data for the patient. The plasma triglyceride concentration was significantly high, and the HDL-cholesterol level was below normal range. Chylomicrons were observed in the patient's plasma after the overnight storage at 4 °C. The patient's plasma apo CII level was not deficient, and apo E genotype was E3/E3. The patient's HbA1c level was normal. In postheparin plasma, the patient's LPL mass was 45 ng/ml, i.e., one fifth of the mean normal value. However, her LPL activity was undetectable (normal: 6.4 ± 2.1 μmol FFA/ml/ h), whereas her HL activity was within normal range (6.6 μmol FFA/ml/h; normal: 8.8 ± 2.9). The direct sequencing analysis showed a G-to-A substitution at nucleotide 818, the second base of codon 188, in exon 5 of the patient's LPL gene. This point mutation replaced glycine (GGG) with glutamic acid (GAG) (G188E). In Ava II RFLP analysis, the patient had a 256-bp fragment alone, demonstrating that the patient was homozygous for the G188E mutation. Homozygosity was compatible with the fact that her parents
were first cousins. These findings allowed us to diagnose the patient as having with LPL deficiency. The ultrasonogram of the carotid arteries indicated that this patient did not exhibit accelerated atherosclerosis. Neither stenosis nor calcification was detected. Ultrasonographic examination of femoral arteries did not detect accelerated atherosclerosis either. The electrocardiogram after exercise-tolerance testing was normal. MDCT angiography for this patient showed a thin atheromatous plaque in the right coronary artery. However, this lesion was consistent with her age and no significant stenosis was detected in any other segments of coronary arteries. These findings indicated that the patient had no clinical signs of accelerated atherosclerosis. 4. Discussion We showed the patient to be homozygous for missense mutation (G188E) in the LPL gene, and diagnosed her with LPL deficiency. Previous reports on patients with G188E have demonstrated that the mutation causes LPL deficiency. In Caucasian, G188E has been shown as one of the prevalent LPL mutations [15,16]. Furthermore, G188E has been detected in LPL deficient patients of non-Caucasian descent. Our patient is the third case with G188E and the eldest in Japan, since 2 patients under 1 y with the same mutation have been reported [17,18]. Patients homozygous for G188E manifested reduced amount of LPL protein without LPL activity [19]. Moreover, in vitro expression studies established that G188E produced mutant LPL protein completely lacking lipolytic activity [20–22]. Our patient had one fifth of the normal LPL protein mass and no LPL activity. These findings were compatible with LPL deficiency with G188E mutation. In complete LPL deficient patients, reports focusing on whether they have accelerated atherosclerosis have been limited (Table 2) [7,8,23–25]. Although chylomicronemia is not believed to be associated with atherosclerosis, this hypothesis has been called into question by Benlian et al. They observed premature atherosclerosis in 4 patients (patients 2–5 in Table 2) and
T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
proposed that defective lipolysis may increase susceptibility to atherosclerosis in humans [7]. Saika et al. observed that a patient with missense mutation L303F suffered from coronary artery disease and severe systemic atherosclerosis (patient 7) [24]. On the other hand, we described a patient homozygous for nonsense mutation who had no evidence of accelerated atherosclerosis (patient 6) [8]. In addition, Kawashiri et al. showed that a patient with another nonsense mutation had no clinically significant atherosclerotic lesions (patient 8) [25]. Accordingly, it has been argued that atherosclerosis among those patients is attributable to the type of LPL mutations [7–9,24,25]. Missense mutation resulting in the presence of LPL protein (even though it has no catalytic activity) develops accelerated atherosclerosis, while nonsense mutations do not accelerate atherosclerosis because of the lack of LPL protein. LPL deficient patient with no mass (patients 6 and 8) had no evidence of accelerated atherosclerosis, and those with LPL mass (patients 2–5, 7) had accelerated atherosclerosis. In contrast to the hypothesis that catalytically inactive LPL protein is pro-atherogenic, our patient homozygous for missense mutation G188E did not have accelerated atherosclerosis. Carotid, femoral, and coronary arteries in the patient were assessed by using ultrasonography and 64-slice MDCT angiography, and no evidence of accelerated atherosclerosis was found. This case suggests that the type of LPL mutation may not be a major factor determining progression of atherosclerosis in LPL deficient patients. Presumably, classic risk factors, such as smoking, hypertension, and glucose intolerance, play a larger role on atherosclerosis than LPL. As far as risk factors are concerned, patients 2–4 with missense mutations had some of risk factors. Patient 3 was a compound heterozygote of G188E and R243C but a smoker, who had angina pectoris. However, patient 8 did not show accelerated atherosclerosis, despite risk factors. Further study is necessary to clarify the impact of LPL mutation types on atherosclerosis in LPL deficient patients. Decreased LDL-cholesterol levels may prevent premature atherosclerosis in LPL deficient patient. LPL plays a key role in production of LDL and the defect of LPL function leads to the impairment of the conversion from VLDL to LDL. Zambon et al. reported a patient who is both heterozygous for FH and homozygous for LPL deficiency and described that the LDL-cholesterol level was significantly lower than the levels of noromolipidemic relatives [26]. The absence of LPL activity lowers plasma LDLcholesterol levels and the susceptibility to atherosclerosis. In summary, the molecular basis of a 60-y-old patient with chylomicronemia has been characterized, and our case suggests that accelerated atherosclerosis may not develop in patients with LPL deficiency, when they have no risk factors.
[10]
Acknowledgement
[19]
This study was supported in part by a Research Grant from the Takeda Science Foundation.
[2] [3]
[4]
[5]
[6]
[7]
[8]
[9]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[20]
References [21] [1] Brunzell J, Deeb S. Familial lipoprotein lipase deficiency, apo C-II deficiency, and hepatic lipase deficiency. In: Scriver C, Beaudet A, Sly W,
103
Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. p. 2789–816. Merkel M, Eckel RH, Goldberg IJ. Lipoprotein lipase: genetics, lipid uptake, and regulation. J Lipid Res 2002;43:1997–2006. Babaev VR, Fazio S, Gleaves LA, Carter KJ, Semenkovich CF, Linton MF. Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo. J Clin Invest 1999;103:1697–705. Wu X, Wang J, Fan J, et al. Localized vessel expression of lipoprotein lipase in rabbits leads to rapid lipid deposition in the balloon-injured arterial wall. Atherosclerosis 2006;187:65–73. Yagyu H, Ishibashi S, Chen Z, et al. Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice. J Lipid Res 1999;40:1677–85. Ebara T, Ramakrishnan R, Steiner G, Shachter NS. Chylomicronemia due to apolipoprotein CIII overexpression in apolipoprotein E-null mice. Apolipoprotein CIII-induced hypertriglyceridemia is not mediated by effects on apolipoprotein E. J Clin Invest 1997;99:2672–81. Benlian P, De Gennes JL, Foubert L, Zhang H, Gagne SE, Hayden M. Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N Engl J Med 1996;335:848–54. Ebara T, Okubo M, Horinishi A, Adachi M, Murase T, Hirano T. No evidence of accelerated atherosclerosis in a 66-yr-old chylomicronemia patient homozygous for the nonsense mutation (Tyr61→stop) in the lipoprotein lipase gene. Atherosclerosis 2001;159:375–9. Foubert L, De Gennes JL, Benlian P, Truffert J, Miao L, Hayden MR. Compound heterozygosity for frameshift mutations in the gene for lipoprotein lipase in a patient with early-onset chylomicronemia. Hum Mutat 1998;Suppl 1:S141–4. Third report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report. Circulation 2002;106:3143–421. Sakaue T, Hirano T, Yoshino G, Sakai K, Takeuchi H, Adachi M. Reactions of direct LDL-cholesterol assays with pure LDL fraction and IDL: comparison of three homogeneous methods. Clin Chim Acta 2000;295:97–106. Murase T, Yamada N, Ohsawa N, Kosaka K, Morita S, Yoshida S. Decline of postheparin plasma lipoprotein lipase in acromegalic patients. Metabolism 1980;29:666–72. Okubo M, Inoue S, Horinishi A, et al. Detection of a new compound heterozygote (del G916/G1401A) for lipoprotein lipase deficiency and a comparative haplotype analysis of the mutant lipoprotein lipase gene from Japanese patients. Atherosclerosis 1999;144:443–7. Hixon JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hha I. J Lipid Res 1990;31:545–8. Bergeron J, Normand T, Bharucha A, et al. Prevalence, geographical distribution and genealogical investigations of mutation 188 of lipoprotein lipase gene in the French Canadian population of Quebec. Clin Genet 1992;41:206–10. Gilbert B, Rouis M, Griglio S, de Lumley L, Laplaud P. Lipoprotein lipase (LPL) deficiency: a new patient homozygote for the preponderant mutation Gly188Glu in the human LPL gene and review of reported mutations: 75% are clustered in exons 5 and 6. Ann Genet 2001;44:25–32. Takagi A, Ikeda Y, Tachi K, Shinozuka T, Yamamoto A. Identification of compound heterozygous mutations (G188E/W382X) of lipoprotein lipase gene in a Japanese infant with hyperchylomicronemia: the G188E mutation was newly identified in Japanese. Clin Chim Acta 1999;285:143–54. Yoshida T, Gotoda T, Okubo M, et al. A Japanese patient with lipoprotein lipase deficiency homozygous for the Gly188Glu mutation prevalent worldwide. J Atheroscler Thromb 2000;7:45–9. Monsalve MV, Henderson H, Roederer G, et al. A missense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries. J Clin Invest 1990;86:728–34. Emi M, Wilson DE, Iverius PH, et al. Missense mutation (Gly–Glu188) of human lipoprotein lipase imparting functional deficiency. J Biol Chem 1990;265:5910–6. Hata A, Ridinger DN, Sutherland SD, et al. Missense mutations in exon 5 of the human lipoprotein lipase gene. Inactivation correlates with loss of dimerization. J Biol Chem 1992;267:20132–9.
104
T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
[22] Peterson J, Ayyobi AF, Ma Y, et al. Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene. J Lipid Res 2002;43:398–406. [23] Hoeg JM, Osborne Jr JC, Gregg RE, Brewer Jr HB. Initial diagnosis of lipoprotein lipase deficiency in a 75-year-old man. Am J Med 1983;75:889–92. [24] Saika Y, Sakai N, Takahashi M, et al. Novel LPL mutation (L303F) found in a patient associated with coronary artery disease and severe systemic atherosclerosis. Eur J Clin Invest 2003;33:216–22.
[25] Kawashiri MA, Higashikata T, Mizuno M, et al. Long-term course of lipoprotein lipase (LPL) deficiency due to homozygous LPL(Arita) in a patient with recurrent pancreatitis, retained glucose tolerance, and atherosclerosis. J Clin Endocrinol Metab 2005;90:6541–4. [26] Zambon A, Torres A, Bijvoet S, et al. Prevention of raised low-density lipoprotein cholesterol in a patient with familial hypercholesterolaemia and lipoprotein lipase deficiency. Lancet 1993;341:1119–21.