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Lipoprotein(a) and the risk of thromboembolism in Thai children
Thrombosis Research 127 (2011) 100–104
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Lipoprotein(a) and the risk of thromboembolism in Thai children Nongnuch Sirachainan a,⁎, Chalermkwan Chaiyong a, Anannit Visudtibhan a, Werasak Sasanakul a, Seksit Osatakul c, Pakawan Wongwerawattanakoon b, Praguywan Kadegasem a, Ampaiwan Chuansumrit a a b c
Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Department of Nursing, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Department of Pediatrics, Faculty of Medicine, Prince of Songkhla University, Hat Yai, Songkhla, Thailand
a r t i c l e
i n f o
Article history: Received 23 March 2010 Received in revised form 10 August 2010 Accepted 1 November 2010 Available online 26 November 2010 Keywords: Lipoprotein(a) Children Thromboembolism
a b s t r a c t High lipoprotein(a) [Lp(a)] level was identified as a risk factor of both venous and arterial thromboembolism (TE), especially in Caucasian children. The Lp(a) level is affected by apo(a) gene. The genetic polymorphisms that associated with Lp(a) level are the size of apo(a) gene, pentanucleotide repeat (TTTTA )n and +93 C/T at promoter region. The increasing size of apo(a) gene, more than 8 pentanucleotide repeats and + 93 C N T polymorphisms are associated with low level of Lp(a) in African and Caucasian populations. This cross sectional, case control study, aims to identify the association of Lp(a) level and the risk for TE in Thai children. Forty-nine patients and 116 healthy children were enrolled. Mean ± SD for age of patients and controls were 7.6 ± 4.7 and 11.2 ± 1.7 years, with female:male ratios of 1:1.2 and 1.8:1, respectively. The median Lp(a) levels in patients was 8.2 (0-87.3) mg/dL and 7.9 (0-74.9) mg/dL in controls, which were not statistically different, P = 0.65. The frequencies of 8 pentanucleotide repeats and + 93 C/T were different compared to Caucasian and African populations but similar to Chinese population. However, both polymorphisms did not affect the level of Lp(a). © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Thromboembolism (TE) is a rare disease in children. It involves both venous and the arterial systems. The incidence of venous thromboembolism (VTE) was around 0.05-0.07 per 10,000 populations per year [1,2] or 5.3 per 10,000 hospital admissions per year. The incidence of TE in Asian populations appears to be lower than Caucasian populations [3]. Our hospital admissions data from 1994 to 2003 revealed an incidence around 3.9 per 10,000 hospital admissions per year [4] and increased to 15.4 per 10,000 hospital admissions per year during 2002 to 2006. The increasing incidence was also reported by another study [5]. The lower incidence of TE may be due to the difference in lifestyles and ethnic background [6]. The risk factors of TE are mainly due to their underlying medical illnesses predisposing to TE accounted for 76% to 98% [7,8]; while the genetic thrombophilia accounted for only 6% to 21% [8,9]. The genetic risk factors in Caucasians are factor V Leiden and prothrombin 20210, accounted for 4.7% to 13% and 2.3% to 3%, respectively [10]; while in Asia, including Thailand, proteins S and C deficiency are more commonly found among patients with TE [11]. Therefore, thrombophilic investigations should be tailored for different region of the world. ⁎ Corresponding author. Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Bangkok, Thailand. Tel.: +662 201 1749; fax: +662 201 1748. E-mail address: [email protected] (N. Sirachainan). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.11.002
Lipoprotein(a) [Lp(a)] consists of low density lipoprotein (LDL), apolipoprotein A [apo(a)], apolipoprotein B [apo(B)], Kringle V-like domain and kringle IV repeats, which have 75% similar structure to plasminogen, thus, interfering with fibrinolytic activity [12]. Other pathophysiologic change is related to atherosclerosis of vessel wall [12–14]. The level of Lp(a) is ranged between 0 - 300 mg/dL and higher in African than those of Caucasian and Asian populations [12,15,16]. The reported studies in children from Caucasian population showed that a high level of Lp(a), more than 30 mg/dL, had the odds ratios (ORs) for venous and arterial TE of 7.2 (95% CI, 3.7 to 14.5) and 7.2 (95% CI,3.8 to 13.8), respectively [17,18]. The meta-analysis study of inherited thrombophilia in children also demonstrated that high levels of Lp(a) increases the risk of VTE [19]. However, some studies did not find an association of high Lp(a) levels in patients with coronary artery disease, VTE or central retinal vein occlusion [20–24]. The level of Lp(a) is determined mainly by genetic polymorphisms. Some acquired conditions can affect the Lp(a) level, such as thyroid disease, inflammatory diseases and children with acute lymphoblastic leukemia treated with L-asparaginase [25–29]. The genetic polymorphisms associated with Lp(a) level are the size of apo(a) gene, pentanucleotide repeat (TTTTA )n and +93 C/T at promoter region. Some reported studies found that a larger size apo(a), 9 pentanucleotide repeats and the change of nucleotide C N T at +93 had lower level of Lp(a) [30–33]. This study aims to document the risk of high Lp(a) and TE in Thai children with TE.
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2. Materials and Methods
2.4. Statistical analysis
2.1. Study populations
The PASW statistics 17.0 (SPSS Inc. Chicago, IL, USA) was used for the analysis. The levels of Lp(a) were expressed as median (range). Mann-Whitney U and Chi-Square tests were used to determine the difference of Lp(a) levels and genetic polymorphisms between patients and controls, respectively. Kruskal-Wallis tests was used to determine the difference of Lp(a) levels among different subgroups. The distribution of Lp(a) levels in patients and controls was compared using percentile distributions. The OR was used to determine the risk of TE with a 95% confidence interval (CI).
The study enrolled Thai children, aged ≤18 years, first diagnosed with either venous or arterial TE, who were treated at either the Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok in the central part; or Prince of Songkhla University, Songkla in the southern part of Thailand during the year 2005-2008. The thrombophilic screenings, including protein C, protein S, antithrombin, homocysteine level, lupus anticoagulant and anticardiolipin antibody, were investigated in patients whose anticoagulant was stopped for one month. The control group consisted of consented healthy Thai children, aged ≤18 years. This study was approved by the institutional review board of the participating institutions. 2.1.1. Inclusion criteria Patients with confirmatory thromboembolic events by computed tomography (CT scan), magnetic resonance imaging (MRI), venography, angiography, or Doppler ultrasound, were enrolled. The time interval before enrollment was at least one month after diagnosis of TE or stopping treatment with anticoagulant. 2.1.2. Exclusion criteria Subjects who received blood component within three months before enrollment and were diagnosed with inflammatory diseases were excluded. Controls, who had family history of thromboembolism at the aged b50 years, were also excluded. 2.1.3. Blood sampling After obtaining informed consent, a 5 ml of blood sample was collected into one EDTA tube and one plain glass tube. Serum was separated by centrifugation at 3,000 rpm for 20 min within 1 h after obtaining the blood sample and then stored at -80 °C until assayed for Lp(a) level. Buffy coat was separated from the EDTA blood sample by the same centrifugation for extracting DNA. 2.2. Lipoprotein(a) assay Lp(a) level was determined by latex-enhanced immunonephelometric assay (Dade Behring , Marburg, Germany). 2.3. Genetic polymorphisms of Pentanucleotide repeat and + 93 C/T DNA was extracted from buffy coat using the conventional phenolchloroform method. 2.3.1. Pentanucleotide repeat polymorphism The DNA amplifications were performed by using the following primers: P1: 5’-TGC GGA AAG ATT GAT ACT ATG C-3’ and P2: 5’-AGG TGG AAG TTG CAG TGA GC-3'as described by Prins et al [34]. The polymerase chain reaction (PCR) products were run on 12% acrylamide gel to identify the size and, then, sent out for sequencing. The sizes of PCR were compared to the number of pentanucleotide repeat [(TTTTA)n] and used as the markers of each pentanucleotide repeat. 2.3.2. +93 C/T polymorphism The DNA amplifications were performed by using the following primers: P1: 5’-AGA TGA AGG TCT AGG GGT GAG-3’ and P2 5’- GAA GAA CCA CTT TAT GTT CC-3’. The polymerase chain reaction products were submitted to restriction digestion with the enzyme Mae II as described by Prins et al [34].
3. Results 3.1. Study population A total of 165 subjects consisting of 49 patients and 116 controls were enrolled. The controls were students at an elemental school with consent from parents to enroll to the study. Among three hundred and forty-five consented subjects, one hundred and sixteen subjects were randomized to the study. The mean age ± SD in patients and controls were 7.6 ± 4.7 and 11.2 ± 1.7 years old, respectively. The proportion of females and males in patients and controls were 1:1.2 and 1.8:1, respectively. Both age (P b 0.01) and gender (P = 0.025) were significantly different. The body mass index (BMI) was recorded in 30 patients and 106 controls; mean ± SD of BMI was 18.7 ± 5.6 in patients and 11.2 ± 1.7 in controls. There was no significant difference of BMI between the two groups (P = 0.075). 3.2. Diagnosis, thrombophilic risk factors and treatment The diagnosis of TE included ischemic stroke (n = 23), deep vein thrombosis (DVT) (n = 5), DVT with pulmonary embolism (n = 3), pulmonary embolism (n = 2), portal vein thrombosis (n = 6), venous sinus thrombosis (n = 6), peripheral arterial thrombosis (n = 3), and thrombus at the VSD patch (n = 1) (Table 1). Five patients were protein C deficient, three hyperhomocysteinemic and two having antiphospholipid antibody. The treatment of thromboembolism was tissue plasminogen activator (tPA), standard and low molecular weight heparin, aspirin, warfarin and fresh frozen plasma in patients with anticoagulant deficiency (Table 1). At the time of study, 11 patients were continued on aspirin while six and four patients were continued on warfarin and LMWH, respectively. 3.3. Lipoprotein(a) level and the risk of thromboembolism The median (range) of Lp(a) levels in patients [8.2 (0-87.3) mg/dL] and in controls [7.9 (0-74.9) mg/dL] were similar (P = 0.65). The patients were classified into three groups; ischemic stroke (n = 23), venous TE (n = 23) and arterial TE (n = 3). The Lp(a) levels in first two groups were also similar when compared to the controls (ATE was excluded from statistical analysis due to the small sample size) (Table 2). At the time of study, level of Lp(a) in patients who received aspirin [6.5 (0-87.3) mg/dL] had no significant difference (P = 0.1) when compared to patients who did not receive aspirin [10.8 (0-54.5) mg/dL]. Further analysis of the risk of TE was performed, the patients whose Lp(a) level was greater than the 75th percentile of 23 mg/dL had the OR of 0.14 (95% CI , 0.01-2.01, P = 0.15) in developing TE. 3.4. Genetic polymorphisms of Pentanucleotide repeat (PNR) and lipoprotein(a) level Forty-five patients and 114 controls had PNR polymorphism testing. The most common PNR polymorphism was 8/8. The incidence
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Table 1 Clinical characteristics of 49 patients. Characteristics
Patients n = 49
Diagnosis Ischemic stroke [n (%)] VTE [n (%)] Portal vein thrombosis [n (%)] Venous sinus thrombosis [n (%)] DVT [n (%)] DVT with PE [n (%)] PE [n (%)] IVC [n (%)] Thrombus at VSD patch [n (%)] ATE [n (%)] Underlying disease Malignancy [n (%)] Autoimmune disease [n (%)] Inflammatory disease [n (%)] Infection [n (%)] Catheter [n (%)] Heart disease [n (%)] Vascular malformation [n (%)] Nephrotic syndrome [n (%)] Down syndrome [n (%)] Cerebral palsy [n (%)] Total [n (%)] Treatment (Acute) Tissue plasminogen activator [n (%)] Standard heparin [n (%)] Low molecular weight heparin [n (%)] Aspirin [n (%)] Warfarin [n (%)] Fresh frozen plasma [n (%)] Total [n (%)]
23 23 6 6 5 3 2 1 1 3
(46.9) (46.9) (12.2) (12.2) (10.2) (6.1) (4.1) (2.0) (2.0) (6.1)
4 3 2 2 2 2 2 1 1 1 20
(8.2) (6.1) (4.1) (4.1) (4.1) (4.1) (4.1) (2.0) (2.0) (2.0) (40.8)
1 1 12 12 3 4 33
(2) (2) (24.5) (24.5) (6.1) (8.2) (67.3)
Abbreviation: ATE, aterial thromboembolism; DVT, deep vein thrombosis; IVC, inferior vena cava; PE, pulmonary embolism; VSD, ventricular septal defect; VTE, venous thromboembolism.
of 8/8 PNR polymorphism in patients and controls were 57.8% and 52.6%, respectively (Table 3). No statistic significance was found of each polymorphism to the level of Lp(a) (Table 4). 3.5. Genetic polymorphisms of +93 C/T and lipoprotein(a) level Forty-five patients and 114 controls had + 93 C/T polymorphism testing. The incidence of +93 C/C polymorphism in patients and controls were 60% and 40%, respectively (Table 3). The polymorphism of + 93 C/T or +93 T/T had no effect on the level of Lp(a) (Table 4). 4. Discussion The present study included 49 patients with both venous and arterial TE and 116 controls. The thrombophilic risk factors were identified in 10 patients (20.4%) including five protein C deficiency, three hyperhomocysteinemia and two antiphospholipid antibody. Although, age and sex in patients and controls were significantly different, the statistical analysis by Pearson correlations coefficient
Table 3 The prevalence of Pentanucleotide repeat and + 93 C/T polymorphisms among patients and controls. Type of polymorphism
Patients [n(%)]
Controls [n(%)]
P-value
Pentanucleotide repeat (PNR) 4/8 5/8 5/9 7/11 8/8 8/9 8/10
0 3 (6.7) 1 (2.2) 0 26 (57.8) 11 (24.4) 3 (6.7)
1 1 1 1 60 34 7
(0.9) (0.9) (0.9) (0.9) (52.6) (30) (6.1)
0.33*
+ 93 C/T CC CT TT C frequency T frequency
29 (60) 16 (40) 0 0.82 0.17
76 (70) 32 (30) 6 (0.05) 0.8 0.19
0.17*
*Chi-Square test evaluating any significant inter-group difference.
showed no correlation of age and sex to the level of Lp(a). This finding was similar to previous reports [35,36]. When compared, the present study found no significant difference of Lp(a) levels in patients and controls. The same results were found when patients were subgrouped into ischemic stroke and VTE. The level of Lp(a) above 75th percentile in our population was 23 mg/dL, which were found in 10.2% of patients and 24% of controls and did not show the increase risk of TE by statistical analysis; while the level of Lp(a) above 75th percentile (30 mg/dL) reported by previous studies was found to have ORs of 7.2 (95% CI, 3.7 to 14.5) for VTE and 7.2 (95% CI 3.8 - 13.8) for ischemic stroke [17,18]. This risk factor in children was also confirmed by the other studies [19,37]. The results from the present study were similar to those of previous studies, i.e. showing no difference of Lp(a) levels between patients with TE including coronary heart disease and controls [20–24]. In addition, the level of Lp(a) in our population was in the low range and closed to the level of Lp(a) reported in Chinese population [15,38]. Other factors affecting Lp(a) level such as inflammation, lipoprotein lipase deficiency and familial hypercholesterolemia were reported [28,39,40]. The limitations of the present study other than the smaller sample size compared to previous studies [17,18] were the only one time testing of Lp(a) level and incomplete data of lipid profiles. Nonetheless, the 29 patients had lipid profiles tested and found no correlation of cholesterol, LDL, high density lipoprotein (HDL), and triglyceride to the Lp(a) levels. Also, aspirin intake in this study did not affect the level of Lp(a) which supported the previous report [41]. The method of measuring Lp(a) level should not affect the result of this study, because the latex-enhanced immunonephelometric assay used in this study is highly correlated to the standard method of radioimmunoassay (correlation coefficient of 0.97) [42].
Table 4 Type of polymorphisms and lipoprotein(a) levels. Type of polymorphism
Table 2 Lipoprotein(a) levels in subgroup of patients with ischemic stroke, venous and arterial thromboembolism compared with controls. Diagnosis
Lipoprotein(a) (mg/dL)
P-value
Normal controls (n = 116) Ischemic stroke (n = 23) Venous thromboembolism (n = 23) Arterial thromboembolism (n = 3)
7.9 (0-74.9) 9.9 (0-54.5) 5.7 (0-87.3) 3.5 (0-7.5)
0.55*
*Kruskal-Wallis test evaluating any significant inter-group difference among Normal controls, Ischemic stroke and Venous thromboembolism groups.
Lipoprotein(a) level (mg/dL)
P-value
Pentanucleotide repeat (PNR) b8 8/8 8/9 N9
7.9 7.3 9.0 10.3
(0-28.1) (0-54.5) (0-87.3) (0-74.8)
0.37*
+ 93 C/T CC CT TT
7.9 (0-74.8) 8.6 (0-87.3) 15.7 (0-41.8)
0.66*
*Kruskal-Wallis test evaluating any significant inter-group difference.
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Two genetic polymorphisms, that have been described to relate to the Lp(a) level including PNRP and + 93 C/T, were studied and it was found that the frequency of 8 PNRP was more common in Thai population than in the Caucasian and the African (allele frequency of 0.72, 0.55 and 0.61, respectively); but the same as Chinese population. In addition, the T allele frequency was more common in Thai population (0.28) than in Caucasian (0.15) and African populations (0.06) [30]. The previous study reported that PNRP more than 8 and +93 C N T polymorphism had lower levels of Lp(a) [31,32]. However, in Thai population, the Lp(a) levels were not different among each PNRP and +93 C/T polymorphism. Therefore, the difference in genetic background may be one of the factors that contributes to the difference result of Lp(a) level and the risk of TE. The Lp(a) level was not significantly affected by age, sex and environmental factors [12,35,43] but mainly by genetic controlling the synthesis of Lp(a) [44]. In the future, other genetic polymorphisms, such as apo(a) size, which was reported to be associated to the level of Lp(a) in Chinese population [38], is warranted. 5. Conclusion Lp(a) level was not significantly different among patients and controls and may not be a risk factor for TE in Thai children. The pentanucleotide repeats and + 93 C/T polymorphisms were not associated with Lp(a) level. Therefore, in the future other genetic polymorphisms should be further identified. Conflicts of Interest The authors indicated no potential conflicts of interest. Acknowledgement The authors are grateful to physicians and paramedical personnel who were involved in taking care of the patients. The authors would like to thank the Thailand Research Fund-Senior Research Scholar 2006 (A.C.) for their support. Nongnuch Sirachainan is a recipient of Career Development Award from Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok. References [1] Andrew M, David M, Adams M, Ali K, Anderson R, Barnard D, et al. Venous thromboembolic complications (VTE) in children: first analyses of the Canadian Registry of VTE. Blood 1994;83(5):1251–7. [2] Chalmers EA. Epidemiology of venous thromboembolism in neonates and children. Thromb Res 2006;118(1):3–12. [3] Cheuk BL, Cheung GC, Cheng SW. Epidemiology of venous thromboembolism in a Chinese population. Br J Surg 2004;91(4):424–8. [4] Sirachainan N, Chuansumrit A, Angchaisuksiri P, Pakakasama S, Hongeng S, Kadegasem P. Venous thromboembolism in Thai children. Pediatr Hematol Oncol 2007;24(4):245–56. [5] Raffini L, Huang YS, Witmer C, Feudtner C. Dramatic increase in venous thromboembolism in children's hospitals in the United States from 2001 to 2007. Pediatrics 2009;124(4):1001–8. [6] Klatsky AL, Baer D. What protects Asians from venous thromboembolism? Am J Med 2004;116(7):493–5. [7] Chuansumrit A, Chiemchanya S, Khowsathit P, Hotrakitya S, Chunharas A, Hathirat P. Thromboembolic complications in Thai pediatric patients. J Med Assoc Thai 2001;84(5):681–7. [8] van Ommen CH, Heijboer H, Buller HR, Hirasing RA, Heijmans HS, Peters M. Venous thromboembolism in childhood: a prospective two-year registry in The Netherlands. J Pediatr 2001;139(5):676–81. [9] Kuhle S, Massicotte P, Chan A, Adams M, Abdolell M, de Veber G, et al. Systemic thromboembolism in children. Data from the 1-800-NO-CLOTS Consultation Service. Thromb Haemost 2004;92(4):722–8. [10] Journeycake JM, Manco-Johnson MJ. Thrombosis during infancy and childhood: what we know and what we do not know. Hematol Oncol Clin North Am 2004;18 (6):1315–38 viii-ix. [11] Anchaisuksiri P. Are Asians genetically differenct from westerners when it comes to VTE? In: Roberts H, editor. Haemophilia and Haemostasis: A case-based approach to management. Malden: Blackwell Publishing; 2007. p. 195–9.
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Lipoprotein(a) and the risk of thromboembolism in Thai children Nongnuch Sirachainan a,⁎, Chalermkwan Chaiyong a, Anannit Visudtibhan a, Werasak Sasanakul a, Seksit Osatakul c, Pakawan Wongwerawattanakoon b, Praguywan Kadegasem a, Ampaiwan Chuansumrit a a b c
Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Department of Nursing, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Department of Pediatrics, Faculty of Medicine, Prince of Songkhla University, Hat Yai, Songkhla, Thailand
a r t i c l e
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Article history: Received 23 March 2010 Received in revised form 10 August 2010 Accepted 1 November 2010 Available online 26 November 2010 Keywords: Lipoprotein(a) Children Thromboembolism
a b s t r a c t High lipoprotein(a) [Lp(a)] level was identified as a risk factor of both venous and arterial thromboembolism (TE), especially in Caucasian children. The Lp(a) level is affected by apo(a) gene. The genetic polymorphisms that associated with Lp(a) level are the size of apo(a) gene, pentanucleotide repeat (TTTTA )n and +93 C/T at promoter region. The increasing size of apo(a) gene, more than 8 pentanucleotide repeats and + 93 C N T polymorphisms are associated with low level of Lp(a) in African and Caucasian populations. This cross sectional, case control study, aims to identify the association of Lp(a) level and the risk for TE in Thai children. Forty-nine patients and 116 healthy children were enrolled. Mean ± SD for age of patients and controls were 7.6 ± 4.7 and 11.2 ± 1.7 years, with female:male ratios of 1:1.2 and 1.8:1, respectively. The median Lp(a) levels in patients was 8.2 (0-87.3) mg/dL and 7.9 (0-74.9) mg/dL in controls, which were not statistically different, P = 0.65. The frequencies of 8 pentanucleotide repeats and + 93 C/T were different compared to Caucasian and African populations but similar to Chinese population. However, both polymorphisms did not affect the level of Lp(a). © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Thromboembolism (TE) is a rare disease in children. It involves both venous and the arterial systems. The incidence of venous thromboembolism (VTE) was around 0.05-0.07 per 10,000 populations per year [1,2] or 5.3 per 10,000 hospital admissions per year. The incidence of TE in Asian populations appears to be lower than Caucasian populations [3]. Our hospital admissions data from 1994 to 2003 revealed an incidence around 3.9 per 10,000 hospital admissions per year [4] and increased to 15.4 per 10,000 hospital admissions per year during 2002 to 2006. The increasing incidence was also reported by another study [5]. The lower incidence of TE may be due to the difference in lifestyles and ethnic background [6]. The risk factors of TE are mainly due to their underlying medical illnesses predisposing to TE accounted for 76% to 98% [7,8]; while the genetic thrombophilia accounted for only 6% to 21% [8,9]. The genetic risk factors in Caucasians are factor V Leiden and prothrombin 20210, accounted for 4.7% to 13% and 2.3% to 3%, respectively [10]; while in Asia, including Thailand, proteins S and C deficiency are more commonly found among patients with TE [11]. Therefore, thrombophilic investigations should be tailored for different region of the world. ⁎ Corresponding author. Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Bangkok, Thailand. Tel.: +662 201 1749; fax: +662 201 1748. E-mail address: [email protected] (N. Sirachainan). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.11.002
Lipoprotein(a) [Lp(a)] consists of low density lipoprotein (LDL), apolipoprotein A [apo(a)], apolipoprotein B [apo(B)], Kringle V-like domain and kringle IV repeats, which have 75% similar structure to plasminogen, thus, interfering with fibrinolytic activity [12]. Other pathophysiologic change is related to atherosclerosis of vessel wall [12–14]. The level of Lp(a) is ranged between 0 - 300 mg/dL and higher in African than those of Caucasian and Asian populations [12,15,16]. The reported studies in children from Caucasian population showed that a high level of Lp(a), more than 30 mg/dL, had the odds ratios (ORs) for venous and arterial TE of 7.2 (95% CI, 3.7 to 14.5) and 7.2 (95% CI,3.8 to 13.8), respectively [17,18]. The meta-analysis study of inherited thrombophilia in children also demonstrated that high levels of Lp(a) increases the risk of VTE [19]. However, some studies did not find an association of high Lp(a) levels in patients with coronary artery disease, VTE or central retinal vein occlusion [20–24]. The level of Lp(a) is determined mainly by genetic polymorphisms. Some acquired conditions can affect the Lp(a) level, such as thyroid disease, inflammatory diseases and children with acute lymphoblastic leukemia treated with L-asparaginase [25–29]. The genetic polymorphisms associated with Lp(a) level are the size of apo(a) gene, pentanucleotide repeat (TTTTA )n and +93 C/T at promoter region. Some reported studies found that a larger size apo(a), 9 pentanucleotide repeats and the change of nucleotide C N T at +93 had lower level of Lp(a) [30–33]. This study aims to document the risk of high Lp(a) and TE in Thai children with TE.
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2. Materials and Methods
2.4. Statistical analysis
2.1. Study populations
The PASW statistics 17.0 (SPSS Inc. Chicago, IL, USA) was used for the analysis. The levels of Lp(a) were expressed as median (range). Mann-Whitney U and Chi-Square tests were used to determine the difference of Lp(a) levels and genetic polymorphisms between patients and controls, respectively. Kruskal-Wallis tests was used to determine the difference of Lp(a) levels among different subgroups. The distribution of Lp(a) levels in patients and controls was compared using percentile distributions. The OR was used to determine the risk of TE with a 95% confidence interval (CI).
The study enrolled Thai children, aged ≤18 years, first diagnosed with either venous or arterial TE, who were treated at either the Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok in the central part; or Prince of Songkhla University, Songkla in the southern part of Thailand during the year 2005-2008. The thrombophilic screenings, including protein C, protein S, antithrombin, homocysteine level, lupus anticoagulant and anticardiolipin antibody, were investigated in patients whose anticoagulant was stopped for one month. The control group consisted of consented healthy Thai children, aged ≤18 years. This study was approved by the institutional review board of the participating institutions. 2.1.1. Inclusion criteria Patients with confirmatory thromboembolic events by computed tomography (CT scan), magnetic resonance imaging (MRI), venography, angiography, or Doppler ultrasound, were enrolled. The time interval before enrollment was at least one month after diagnosis of TE or stopping treatment with anticoagulant. 2.1.2. Exclusion criteria Subjects who received blood component within three months before enrollment and were diagnosed with inflammatory diseases were excluded. Controls, who had family history of thromboembolism at the aged b50 years, were also excluded. 2.1.3. Blood sampling After obtaining informed consent, a 5 ml of blood sample was collected into one EDTA tube and one plain glass tube. Serum was separated by centrifugation at 3,000 rpm for 20 min within 1 h after obtaining the blood sample and then stored at -80 °C until assayed for Lp(a) level. Buffy coat was separated from the EDTA blood sample by the same centrifugation for extracting DNA. 2.2. Lipoprotein(a) assay Lp(a) level was determined by latex-enhanced immunonephelometric assay (Dade Behring , Marburg, Germany). 2.3. Genetic polymorphisms of Pentanucleotide repeat and + 93 C/T DNA was extracted from buffy coat using the conventional phenolchloroform method. 2.3.1. Pentanucleotide repeat polymorphism The DNA amplifications were performed by using the following primers: P1: 5’-TGC GGA AAG ATT GAT ACT ATG C-3’ and P2: 5’-AGG TGG AAG TTG CAG TGA GC-3'as described by Prins et al [34]. The polymerase chain reaction (PCR) products were run on 12% acrylamide gel to identify the size and, then, sent out for sequencing. The sizes of PCR were compared to the number of pentanucleotide repeat [(TTTTA)n] and used as the markers of each pentanucleotide repeat. 2.3.2. +93 C/T polymorphism The DNA amplifications were performed by using the following primers: P1: 5’-AGA TGA AGG TCT AGG GGT GAG-3’ and P2 5’- GAA GAA CCA CTT TAT GTT CC-3’. The polymerase chain reaction products were submitted to restriction digestion with the enzyme Mae II as described by Prins et al [34].
3. Results 3.1. Study population A total of 165 subjects consisting of 49 patients and 116 controls were enrolled. The controls were students at an elemental school with consent from parents to enroll to the study. Among three hundred and forty-five consented subjects, one hundred and sixteen subjects were randomized to the study. The mean age ± SD in patients and controls were 7.6 ± 4.7 and 11.2 ± 1.7 years old, respectively. The proportion of females and males in patients and controls were 1:1.2 and 1.8:1, respectively. Both age (P b 0.01) and gender (P = 0.025) were significantly different. The body mass index (BMI) was recorded in 30 patients and 106 controls; mean ± SD of BMI was 18.7 ± 5.6 in patients and 11.2 ± 1.7 in controls. There was no significant difference of BMI between the two groups (P = 0.075). 3.2. Diagnosis, thrombophilic risk factors and treatment The diagnosis of TE included ischemic stroke (n = 23), deep vein thrombosis (DVT) (n = 5), DVT with pulmonary embolism (n = 3), pulmonary embolism (n = 2), portal vein thrombosis (n = 6), venous sinus thrombosis (n = 6), peripheral arterial thrombosis (n = 3), and thrombus at the VSD patch (n = 1) (Table 1). Five patients were protein C deficient, three hyperhomocysteinemic and two having antiphospholipid antibody. The treatment of thromboembolism was tissue plasminogen activator (tPA), standard and low molecular weight heparin, aspirin, warfarin and fresh frozen plasma in patients with anticoagulant deficiency (Table 1). At the time of study, 11 patients were continued on aspirin while six and four patients were continued on warfarin and LMWH, respectively. 3.3. Lipoprotein(a) level and the risk of thromboembolism The median (range) of Lp(a) levels in patients [8.2 (0-87.3) mg/dL] and in controls [7.9 (0-74.9) mg/dL] were similar (P = 0.65). The patients were classified into three groups; ischemic stroke (n = 23), venous TE (n = 23) and arterial TE (n = 3). The Lp(a) levels in first two groups were also similar when compared to the controls (ATE was excluded from statistical analysis due to the small sample size) (Table 2). At the time of study, level of Lp(a) in patients who received aspirin [6.5 (0-87.3) mg/dL] had no significant difference (P = 0.1) when compared to patients who did not receive aspirin [10.8 (0-54.5) mg/dL]. Further analysis of the risk of TE was performed, the patients whose Lp(a) level was greater than the 75th percentile of 23 mg/dL had the OR of 0.14 (95% CI , 0.01-2.01, P = 0.15) in developing TE. 3.4. Genetic polymorphisms of Pentanucleotide repeat (PNR) and lipoprotein(a) level Forty-five patients and 114 controls had PNR polymorphism testing. The most common PNR polymorphism was 8/8. The incidence
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Table 1 Clinical characteristics of 49 patients. Characteristics
Patients n = 49
Diagnosis Ischemic stroke [n (%)] VTE [n (%)] Portal vein thrombosis [n (%)] Venous sinus thrombosis [n (%)] DVT [n (%)] DVT with PE [n (%)] PE [n (%)] IVC [n (%)] Thrombus at VSD patch [n (%)] ATE [n (%)] Underlying disease Malignancy [n (%)] Autoimmune disease [n (%)] Inflammatory disease [n (%)] Infection [n (%)] Catheter [n (%)] Heart disease [n (%)] Vascular malformation [n (%)] Nephrotic syndrome [n (%)] Down syndrome [n (%)] Cerebral palsy [n (%)] Total [n (%)] Treatment (Acute) Tissue plasminogen activator [n (%)] Standard heparin [n (%)] Low molecular weight heparin [n (%)] Aspirin [n (%)] Warfarin [n (%)] Fresh frozen plasma [n (%)] Total [n (%)]
23 23 6 6 5 3 2 1 1 3
(46.9) (46.9) (12.2) (12.2) (10.2) (6.1) (4.1) (2.0) (2.0) (6.1)
4 3 2 2 2 2 2 1 1 1 20
(8.2) (6.1) (4.1) (4.1) (4.1) (4.1) (4.1) (2.0) (2.0) (2.0) (40.8)
1 1 12 12 3 4 33
(2) (2) (24.5) (24.5) (6.1) (8.2) (67.3)
Abbreviation: ATE, aterial thromboembolism; DVT, deep vein thrombosis; IVC, inferior vena cava; PE, pulmonary embolism; VSD, ventricular septal defect; VTE, venous thromboembolism.
of 8/8 PNR polymorphism in patients and controls were 57.8% and 52.6%, respectively (Table 3). No statistic significance was found of each polymorphism to the level of Lp(a) (Table 4). 3.5. Genetic polymorphisms of +93 C/T and lipoprotein(a) level Forty-five patients and 114 controls had + 93 C/T polymorphism testing. The incidence of +93 C/C polymorphism in patients and controls were 60% and 40%, respectively (Table 3). The polymorphism of + 93 C/T or +93 T/T had no effect on the level of Lp(a) (Table 4). 4. Discussion The present study included 49 patients with both venous and arterial TE and 116 controls. The thrombophilic risk factors were identified in 10 patients (20.4%) including five protein C deficiency, three hyperhomocysteinemia and two antiphospholipid antibody. Although, age and sex in patients and controls were significantly different, the statistical analysis by Pearson correlations coefficient
Table 3 The prevalence of Pentanucleotide repeat and + 93 C/T polymorphisms among patients and controls. Type of polymorphism
Patients [n(%)]
Controls [n(%)]
P-value
Pentanucleotide repeat (PNR) 4/8 5/8 5/9 7/11 8/8 8/9 8/10
0 3 (6.7) 1 (2.2) 0 26 (57.8) 11 (24.4) 3 (6.7)
1 1 1 1 60 34 7
(0.9) (0.9) (0.9) (0.9) (52.6) (30) (6.1)
0.33*
+ 93 C/T CC CT TT C frequency T frequency
29 (60) 16 (40) 0 0.82 0.17
76 (70) 32 (30) 6 (0.05) 0.8 0.19
0.17*
*Chi-Square test evaluating any significant inter-group difference.
showed no correlation of age and sex to the level of Lp(a). This finding was similar to previous reports [35,36]. When compared, the present study found no significant difference of Lp(a) levels in patients and controls. The same results were found when patients were subgrouped into ischemic stroke and VTE. The level of Lp(a) above 75th percentile in our population was 23 mg/dL, which were found in 10.2% of patients and 24% of controls and did not show the increase risk of TE by statistical analysis; while the level of Lp(a) above 75th percentile (30 mg/dL) reported by previous studies was found to have ORs of 7.2 (95% CI, 3.7 to 14.5) for VTE and 7.2 (95% CI 3.8 - 13.8) for ischemic stroke [17,18]. This risk factor in children was also confirmed by the other studies [19,37]. The results from the present study were similar to those of previous studies, i.e. showing no difference of Lp(a) levels between patients with TE including coronary heart disease and controls [20–24]. In addition, the level of Lp(a) in our population was in the low range and closed to the level of Lp(a) reported in Chinese population [15,38]. Other factors affecting Lp(a) level such as inflammation, lipoprotein lipase deficiency and familial hypercholesterolemia were reported [28,39,40]. The limitations of the present study other than the smaller sample size compared to previous studies [17,18] were the only one time testing of Lp(a) level and incomplete data of lipid profiles. Nonetheless, the 29 patients had lipid profiles tested and found no correlation of cholesterol, LDL, high density lipoprotein (HDL), and triglyceride to the Lp(a) levels. Also, aspirin intake in this study did not affect the level of Lp(a) which supported the previous report [41]. The method of measuring Lp(a) level should not affect the result of this study, because the latex-enhanced immunonephelometric assay used in this study is highly correlated to the standard method of radioimmunoassay (correlation coefficient of 0.97) [42].
Table 4 Type of polymorphisms and lipoprotein(a) levels. Type of polymorphism
Table 2 Lipoprotein(a) levels in subgroup of patients with ischemic stroke, venous and arterial thromboembolism compared with controls. Diagnosis
Lipoprotein(a) (mg/dL)
P-value
Normal controls (n = 116) Ischemic stroke (n = 23) Venous thromboembolism (n = 23) Arterial thromboembolism (n = 3)
7.9 (0-74.9) 9.9 (0-54.5) 5.7 (0-87.3) 3.5 (0-7.5)
0.55*
*Kruskal-Wallis test evaluating any significant inter-group difference among Normal controls, Ischemic stroke and Venous thromboembolism groups.
Lipoprotein(a) level (mg/dL)
P-value
Pentanucleotide repeat (PNR) b8 8/8 8/9 N9
7.9 7.3 9.0 10.3
(0-28.1) (0-54.5) (0-87.3) (0-74.8)
0.37*
+ 93 C/T CC CT TT
7.9 (0-74.8) 8.6 (0-87.3) 15.7 (0-41.8)
0.66*
*Kruskal-Wallis test evaluating any significant inter-group difference.
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Two genetic polymorphisms, that have been described to relate to the Lp(a) level including PNRP and + 93 C/T, were studied and it was found that the frequency of 8 PNRP was more common in Thai population than in the Caucasian and the African (allele frequency of 0.72, 0.55 and 0.61, respectively); but the same as Chinese population. In addition, the T allele frequency was more common in Thai population (0.28) than in Caucasian (0.15) and African populations (0.06) [30]. The previous study reported that PNRP more than 8 and +93 C N T polymorphism had lower levels of Lp(a) [31,32]. However, in Thai population, the Lp(a) levels were not different among each PNRP and +93 C/T polymorphism. Therefore, the difference in genetic background may be one of the factors that contributes to the difference result of Lp(a) level and the risk of TE. The Lp(a) level was not significantly affected by age, sex and environmental factors [12,35,43] but mainly by genetic controlling the synthesis of Lp(a) [44]. In the future, other genetic polymorphisms, such as apo(a) size, which was reported to be associated to the level of Lp(a) in Chinese population [38], is warranted. 5. Conclusion Lp(a) level was not significantly different among patients and controls and may not be a risk factor for TE in Thai children. The pentanucleotide repeats and + 93 C/T polymorphisms were not associated with Lp(a) level. Therefore, in the future other genetic polymorphisms should be further identified. Conflicts of Interest The authors indicated no potential conflicts of interest. Acknowledgement The authors are grateful to physicians and paramedical personnel who were involved in taking care of the patients. The authors would like to thank the Thailand Research Fund-Senior Research Scholar 2006 (A.C.) for their support. Nongnuch Sirachainan is a recipient of Career Development Award from Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok. References [1] Andrew M, David M, Adams M, Ali K, Anderson R, Barnard D, et al. Venous thromboembolic complications (VTE) in children: first analyses of the Canadian Registry of VTE. Blood 1994;83(5):1251–7. [2] Chalmers EA. Epidemiology of venous thromboembolism in neonates and children. Thromb Res 2006;118(1):3–12. [3] Cheuk BL, Cheung GC, Cheng SW. Epidemiology of venous thromboembolism in a Chinese population. Br J Surg 2004;91(4):424–8. [4] Sirachainan N, Chuansumrit A, Angchaisuksiri P, Pakakasama S, Hongeng S, Kadegasem P. Venous thromboembolism in Thai children. Pediatr Hematol Oncol 2007;24(4):245–56. [5] Raffini L, Huang YS, Witmer C, Feudtner C. Dramatic increase in venous thromboembolism in children's hospitals in the United States from 2001 to 2007. Pediatrics 2009;124(4):1001–8. [6] Klatsky AL, Baer D. What protects Asians from venous thromboembolism? Am J Med 2004;116(7):493–5. [7] Chuansumrit A, Chiemchanya S, Khowsathit P, Hotrakitya S, Chunharas A, Hathirat P. Thromboembolic complications in Thai pediatric patients. J Med Assoc Thai 2001;84(5):681–7. [8] van Ommen CH, Heijboer H, Buller HR, Hirasing RA, Heijmans HS, Peters M. Venous thromboembolism in childhood: a prospective two-year registry in The Netherlands. J Pediatr 2001;139(5):676–81. [9] Kuhle S, Massicotte P, Chan A, Adams M, Abdolell M, de Veber G, et al. Systemic thromboembolism in children. Data from the 1-800-NO-CLOTS Consultation Service. Thromb Haemost 2004;92(4):722–8. [10] Journeycake JM, Manco-Johnson MJ. Thrombosis during infancy and childhood: what we know and what we do not know. Hematol Oncol Clin North Am 2004;18 (6):1315–38 viii-ix. [11] Anchaisuksiri P. Are Asians genetically differenct from westerners when it comes to VTE? In: Roberts H, editor. Haemophilia and Haemostasis: A case-based approach to management. Malden: Blackwell Publishing; 2007. p. 195–9.
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