Pathogenesis and Host Response
Association of CPT II Gene With Risk of Acute Encephalitis in Chinese Children Jianxia Hu, MD,* Zongbo Chen, MD,† Xiaoyi Liu, MD,‡ Zhihong Chen, MD,† Dandan Xin, MD,† and Peipei Liu, MD† Background: Mutations of the CPT II gene cause CPT II deficiency, an inborn metabolic error affecting mitochondrial fatty acid β-oxidation. Associations and mechanisms of CPT II gene with acute encephalitis need to be elucidated. We aimed to investigate the associations of CPT II gene variants and CPT II activity with development of acute encephalitis. Methods: A total of 440 blood-unrelated Chinese children with acute encephalitis and 229 healthy controls were enrolled in this case control study. Sequencing of 5 exons of the CPT II gene was carried out to look for the variants associated with acute encephalitis. CPT II activity and blood adenosine triphosphate concentration were examined during high fever and convalescent phase to confirm the hypothesis. Results: Polymorphism of rs2229291 in CPT II gene was significantly associated with an increased risk of acute encephalitis (P = 0.031), where as rs1799821 displayed a decrease risk (P = 0.018). Positive association was found between rs2229291 and patients with fever at onset of seizure and degree of pathogenetic condition (P = 0.018 and P = 0.023), but not for rs1799821. CPT II activity of patients with rs2229291 reduced greatly during high fever compared with the convalescent phase. Conclusions: rs2229291 and rs1799821 variants in CPT II gene might be 1 of the predisposing factors of acute encephalitis. Key Words: carnitine palmitoyl transferase II, acute encephalitis, polymorphisms (Pediatr Infect Dis J 2014;33:1077–1082)
childhood in East Asians. Chen et al9 discovered a significant portion of patients who had severe influenza-associated encephalopathy (IAE) and showed abnormal acylcarnitine patterns indicating a failing mitochondrial fatty acid oxidation. Shinohara et al10 confirmed these findings in 29 Japanese patients with acute encephalopathy. Also, some reports have presumed that genotypic variants of CPT II gene are associated with fatal or severe cases of IAE.11–14 Mutations of the CPT II gene cause CPT II deficiency, an inborn metabolic error affecting mitochondrial fatty acid β-oxidation. When patients with CPT II deficiency are infected with viruses, some develop energy failure and show a clinical course resembling that of acute encephalopathy.15 There is a great density of mitochondria to supply constant adenosine triphosphate (ATP) in the brain capillary endothelium which can maintain the physiologic functions of significant active transport mechanisms, electrochemical gradients, autoregulatory adjustments and regulation of tight junctional complexes. CPT II deficiency might cause lower blood ATP values in the acute phase of encephalopathy during high fever. Thus the blood brain barrier (BBB) breakdown may occur at an initial stage of encephalopathy under the condition of ATP reduction, thus leading to subsequent brain edema.16 However, there are few reports about the mechanisms and associations between CPT II gene variants and acute encephalitis. In the present study, we investigated the associations and possible mechanisms of CPT II gene variants with acute encephalitis.
MATERIALS AND METHODS
C
arnitine palmitoyl transferase II (CPT II) is a catalytically active, malonyl CoA insensitive enzyme localized on the mitochondrial inner membrane and plays a key role in the transport of long-chain fatty acids into the mitochondria. CPT II facilitates the β-oxidation of long-chain fatty acids in the mitochondria and is ubiquitously expressed in all tissues that require fatty acid oxidation as an energy-producing pathway.1,2 CPT II deficiency can induce a disorder of long-chain fatty acids oxidation and impaired energy production, the effects of which are most pronounced during fasting or infection, when fatty acid breakdown is an essential energy source.3,4 Acute encephalitis is an acute brain dysfunction that usually occurs at the early stage of infectious diseases with symptoms of high fever, impaired consciousness, convulsions or seizures associated with brain edema.5,6 The antecedent infection of acute encephalitis is usually viral, such as human enterovirus, human herpes virus-6 and some other viruses,7–9 and its incidence is highest in infancy and early Accepted for publication March 22, 2014. From the *Stem Cell Center; †Department of Paediatrics; and ‡Department of Galactophore, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, China. The authors have no funding or conflicts of interest to disclose. Address for correspondence: Zongbo Chen, Department of Paediatrics, the Affiliated Hospital of Medical College, Qingdao University, No. 59, Haier Road, Qingdao 266000, China. E-mail: [email protected]. Copyright © 2014 by Lippincott Williams & Wilkins ISSN: 0891-3668/14/3310-1077 DOI: 10.1097/INF.0000000000000368
Patients The study protocol was approved by the Ethical Committee of the Affiliated Hospital of the Medical College, Qingdao University. Informed consent according to the Declaration of Helsinki was provided by every patient’s guardian. Between May 2010 and November 2012, 600 patients were screened for enrollment in the Department of Pediatrics of our hospital. Of these patients, 82 patients were excluded for diagnosis as bacterial encephalitis, 518 patients fitted the inclusion criteria and were personally interviewed. After that, 440 patients with acute encephalitis were enrolled whereas other 78 patients refused to participate in the study. All patients were diagnosed as viral encephalitis according to the clinical manifestations and laboratory examinations of the disease. Patients with the abrupt onset of febrile convulsive status epilepticus and cataphora were considered as severe degree of pathogenetic condition. Patients with the abrupt onset of febrile convulsive status epilepticus but not with consciousness disorder were considered as mild degree of pathogenetic condition. Viral nucleic acids from blood, feces and cerebrospinal fluid were examined by reverse transcription-polymerase chain reaction (PCR). Patients with other metabolism disorders, cardiopathy, dysgnosia, hypoplasia or cerebral palsy caused by innateness, trauma or other nonviral infections were excluded.
Controls Two-hundred and twenty-nine healthy children volunteers with a similar history of virus infection but without history of encephalitis, including 112 male and 117 female, at 2–8 years of
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age, were enrolled as controls. The conventional treatment for controls was rest, drinking more water, vitamin supplement and antiviral therapy (eg, Rimantadine). Volunteers with other metabolism disorders, cardiopathy, dysgnosia, congenital hypoplasia or cerebral palsy, trauma or other nonviral infections were excluded. All their previous histories were carefully recorded.
Clinical Data Analysis For all subjects, patients and controls, during the treatment, the hyperthermia, duration of fever, blood routine, electroencephalogram, cerebrospinal fluid routine, computerized tomography or magnetic resonance imaging (MRI) of brain and electrocardiogram were all examined and carefully recorded. All symptoms including headache, nausea, vomit, lethargy, clouding of consciousness, cataphora, convulsion, meningeal irritation sign, ataxia, respiratory or circulatory failure, abdominal pain, diarrhea, rash, arthralgia and pharyngalgia were recorded carefully.
Genomic DNA Extraction Genomic DNA was extracted from peripheral blood using a commercial blood Genomic DNA extraction kit (from Dongsheng Biotech, Beijing, China) according to the manufacturer’s instructions. The extracted DNA samples were stored in a freezer at −80°C until genetic polymorphism analyses were performed.
Genotyping Genomic DNA from whole blood was evaluated by electrophoresis (1% agarose) and diluted to a working concentration of 5–10 ng/µL. PCR of 5 exons of the CPT II gene was carried out with intron-based primers in genomic DNA. For P2F1/R1 segment of CPT II gene, the reaction condition was as following: a 10 µL mixture was prepared for each reaction and included 1× GC buffer, 0.2 mmol/L dNTP, 0.2 µmol/L of each primer, 0.25 units Ex Taq polymerase (Takara, Dalian, China) and 1 µL template DNA. The cycling program was 95°C for 5 minutes; 35 cycles of 96°C for 10 seconds, 68°C for 1 minute per cycle and 72°C for 2 minutes. For other segments, the reaction condition was as following: a 10 µL mixture was prepared for each reaction and included 1× Ex Taq buffer, 2.0 mmol/L Mg2+, 0.2 mmol/L dNTP, 0.2 µmol/L of each primer, 0.25 units Ex Taq polymerase and 1 µL template DNA. The cycling program was 95°C for 5 minutes; 11 cycles of 94°C for 15 seconds, 62°C for 40 seconds per cycle, 72°C for 1 minute; 24 cycles of 94°C for 15 seconds, 56°C for 30 seconds, 72°C for 1 minute and 72°C for 2 minutes. The sequences of the PCR products were analyzed with the ABI BigDye 3.1 Sequencing Kit on an ABI 3130 XL Genetic Analyzer (Applied Biosystems Inc, Foster City, CA). Reaction mixture included 2 µL BigDye3.1 mix, 2 µL sequencing primer (0.4 µmol/L) and 1–2 µL purified PCR product. The cycling program was 96°C for 1 minute; 28 cycles of 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes. Each PCR product was sequenced in both strands, and all analyses were performed at least twice independently.
Preparation of Subjects’ Lymphoblasts and Culture Blood samples (2 mL) were obtained from subjects, patients and controls, by venipuncture into a sterile EDTA blood collection tube. Lymphocytes were separated from peripheral blood, diluted (1:1, v/v) with sterile saline, by centrifugation (800 g, 20 minutes) over 2 mL of Lymphoprep (Nycomed, Denmark). The lymphocyte layer was recovered and washed twice with phosphate-buffered saline by centrifugation at 250 g for 10 minutes each, and then maintained in PRMI-1640 (GIBCO) supplemented with 12.5% fetal calf serum. Cells were incubated with 5% CO2 at 37°C for
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7 days. Lymphoblastic cell lines were established by infecting peripheral blood lymphocytes with the Epstein Barr virus. Cells were grown in suspension in an SC flask (Greiner 658190, Germany) in an upright position, in 10 mL of PRMI-1640 medium that contained 12.5% fetal calf serum, maintained at 37°C. Fluid was routinely changed every 2 days by removing the medium above the settled cells and replacing it with an equal volume of fresh medium.
Analysis of CPT II Activity CPT II activities of subjects’ lymphoblasts were analyzed as previously described.13 To prepare whole cell extracts, cells were harvested and washed twice with phosphate-buffered saline at 250 g for 10 minutes and then lysed with 0.5 mL of ice-cold lysis buffer (5 mmol/L Tris-HCl buffer, pH 7.4, containing 1% Tween-20 and 0.5 mol/L KCl), then centrifuged at 147,600 g for 1 hour at 4°C. CPT II activities in the lysates of lymphoblasts were measured at 30°C for 2 hours, by detection of the palmitoyl-L-[methyl-3 H] carnitine formed from 200 mmol/L L-[methyl-3 H] carnitine and 50 mmol/L palmitoyl-CoA. The formed palmitoyl-L-[methyl-3H] carnitine was extracted with 1-butanol and radioactivity was counted by a liquid scintillation counter (Model LS 6500; Beckman, Fullerton). To analyze the heat stability of CPT II, cell lysates were preincubated at 35, 37, 39 and 41°C for 0–120 minutes, and enzyme activities were measured by addition of 200 mmol/L L-[methyl-3H] carnitine followed by incubation at 30°C for 2 hours. Protein concentrations in the cell lysates were measured using the BCATM Protein Assay Kit (Thermo SCIENTIFIC, Pittsburgh, PA).
Measurement of Blood ATP Concentrations ATP concentrations in whole blood lysates were measured by an ENLITEN ATP assay system bioluminescence detection kit (Promega, Madison, WI) according to the instructions provided by the manufacturer and the values were expressed as ATP levels in whole blood. Briefly, 0.5% trichloroacetic acid was added in blood for ATP efficient release. Then, 25 mmol/L Tris-acetate (pH 7.75) was used for neutralization. After addition of recombinant luciferase/luciferin reagent (rL/L), luminescence was measured using a 10-second integration time with a microplate luminometer (Lmax) and SOFTmax PRO software (Molecular Devices) and was normalized to protein concentration. The ATP standard curve was generated using the ATP standard (10−7 mol/L) included in the kit.
Statistical Analysis All statistical analyses were conducted using Prism 5. The observed genotype frequencies in controls were tested for the Hardy-Weinberg equilibrium using the χ2 test. Associations between gene variants and risk of acute encephalitis were tested using the χ2 test and odds ratios (OR) and 95% confidence intervals (95% CI) were calculated. Normally distributed, continuous measures are presented as mean ± SD. The differences of CPT II activities and blood ATP levels between high fever and convalescent phase were tested by independent samples t test and paired samples t test. We initially considered all P values < 0.05 as nominally significant but further assessed the significance using the conservative Bonferroni correction to account for multiple comparisons. All P values were subjected to Bonferroni correction for multiple testing by multiplying uncorrected P values by the number of independent tests performed (8 for single nucleotide polymorphism testing). We presented the corrected P values. A difference with a corrected value of P < 0.05 was considered significant.
RESULTS A total of 629 subjects were enrolled in this study, including 440 acute encephalitis patients and 229 controls. All these subjects © 2014 Lippincott Williams & Wilkins
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TABLE 1. Clinical Summary of All Subjects Patients (n = 440)
Variable Age (year) Age below 3 years [n (%)] Gender [n (%)] Girl Boy Pathogen Diagnosis Duration of high fever (day) Hyperthermia (°C) Duration of seizure (min) Degree of pathogenetic condition [n (%)] Mild Severe Electroencephalogram (abnormal) [n (%)] Brain MRI (abnormal) [n (%)] Prognosis [n (%)] Good Poor
Controls (n = 229)
3.9 ± 1.8 303 (68.9%)
4.6 ± 2.0 163 (71.2%)
227 (51.6%) 213 (48.4%) Virus Acute encephalitis 2.3 ± 1.9 38.3 ± 3.1 15.6 ± 10.2
117 (51.1%) 112 (48.9%)
365 (83.0%) 75 (17.0%) 362 (82.3%) 289 (65.7%) 399 (90.7%) 41 (9.3%)
Data of age, duration of high fever, hyperthermia, and duration of seizure are presented as mean ± SD.
are Chinese Han people. Clinical parameters of all subjects are shown in Table 1. About 70% of patients were 0.99). Table 2 showed the risks of acute encephalitis in relation to the 2 CPT II variants. The proportions of the TT, TG and GG genotypes of rs2229291 in controls and patients were 65.1%, 28.4% and 6.5% and 54.5%, 37.7% and 7.8%, respectively, whereas, for rs1799821, the proportions of the AA, AG and GG genotypes in controls and patients were 47.6%, 41.0% and 11.4% and 55.0%, 39.3% and 5.7%, respectively. There were statistical differences in the 2 polymorphisms between patients and controls (P = 0.031 and P = 0.018). As shown in Table 2, the TG+GG genotypes of rs2229291 were markedly overrepresented in the patients compared with controls (P = 0.009 with Bonferroni correction), whereas GG genotype of rs1799821 was significantly lower in patients compared with controls (P = 0.005 with Bonferroni correction). By respectively using the TT and AA genotype as reference, significant associations were found between the TG (rs2229291) and GG (rs1799821) genotypes and the risk of acute encephalitis (OR = 1.346 and OR = 2.309, respectively; P = 0.010 and P = 0.005 with Bonferroni correction). After Bonferroni adjustment, there were statistically significant associations between rs1799821 G allele (P = 0.011, OR = 1.379, 95% CI: 1.075–1.767) and acute encephalitis. Furthermore, no statistical differences in compound heterozygotes of rs2229291 and rs1799821 were found between patients and controls (P = 0.697).
Associations of rs2229291 and rs1799821 With Clinical Characteristics To investigate whether the genotypes relate to different clinical characteristics, including temperature at onset of seizure (≤39°C or >39°C), degree of pathogenetic condition (mild or severe), electroencephalogram (normal or abnormal), brain MRI (normal or abnormal) and prognosis (good or poor), we compared genotype distributions between patients with these clinical characteristics. Significant differences were found in the TG+GG genotypic frequencies of rs2229291 between patients with temperature at onset of seizure (≤ 39°C), degree of pathogenetic condition (severe; P =0.018 and P=0.023 with Bonferroni correction, showed in Table 3). No significant associations were found between rs1799821 and clinical characteristics.
TABLE 2. The Genotype and Allele Distributions of rs2229291 and rs1799821 in CPT II Gene Between Patients and Controls Polymorphism rs2229291 Genotypes TT TG GG TG+GG Alleles T G rs1799821 Genotypes AA AG GG AG + GG Alleles A G
Patients [n(%)]
Controls [n(%)]
χ2
OR
95% CI
240 (54.5%) 166 (37.7%) 34 (7.8%) 200 (45.5%)
149 (65.1%) 65 (28.4%) 15 (6.5%) 80 (34.9%)
0.010 0.294 0.009
1.346 0.711 1.301
1.065–1.701 0.374–1.349 1.061–1.596
646 (73.4%) 234 (26.6%)
363 (79.3%) 95 (20.7%)
0.018
1.246
1.032–1.504
242 (55.0%) 173 (39.3%) 25 (5.7%) 198 (45.0%)
109 (47.6%) 94 (41.0%) 26 (11.4%) 120 (52.4%)
0.276 0.005 0.069
0.882 2.309 0.823
0.704–1.105 1.275–4.181 0.667–1.016
657 (74.7%) 223 (25.3%)
312 (68.1%) 146 (31.9%)
0.011
1.379
1.075–1.767
Tests of independence between gene variants and risk of acute encephalitis were tested using the χ2 test. OR and 95% CI provide the association between gene variants and WT and group (acute encephalitis vs. controls).
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TABLE 3. The Association Between rs2229291 and Clinical Characteristics of Patients Parameters
TT [n (%)]
Temperature at onset of seizure ≤39°C 93 (48.2%) >39°C 147 (59.5%) Degree of pathogenetic condition Mild 208 (57.0%) Severe 32 (42.7%) Electroencephalogram Normal 42 (53.8%) Abnormal 198(54.7%) Brain MRI Normal 92 (60.9%) Abnormal 148 (51.2%) Prognosis Good 215 (53.9%) Poor 25 (61.0%)
TG + GG [n(%)]
χ2
OR
95% CI
100 (51.8%) 100 (40.5%)
0.018
1.280
1.044–1.568
157 (43.0%) 43 (57.3%)
0.023
1.780
1.077–2.942
36 (46.2%) 164 (45.3%)
0.891
0.966
0.591–1.579
59 (39.1%) 141 (48.8%)
0.052
0.801
0.635–1.010
184 (46.1%) 16 (39.0%)
0.385
0.748
0.387–1.443
Tests of independence between gene variants and clinical characteristics of acute encephalitis were tested using the χ2 test. OR and 95% CI provide the association between gene variants and clinical characteristics.
Lymphocyte CPT II Activity in the Patients All patients with rs2229291 variant (n = 200) showed significant reductions of CPT II activity at 41°C (average slip 35 ± 18.3%, P = 0.001). As shown in Fig. 1, CPT II activity using peripheral lymphocytes of a patient with rs2229291 was significantly reduced to about 60% during incubation for 120 minutes at 41°C compared with those at 35°C and 37°C. CPT II activities in patients with rs1799821 variant (n = 198) were not reduced obviously during incubation for 120 min at 41°C compared with that at 35°C (average slip 10 ± 5.9%, P = 0.13).
the CPT II variants may cause mitochondrial fuel usage failure during high fever in the brain endothelial cells. Mutations of the CPT II gene cannot cause low rates of protein synthesis but can cause a severe reduction of CPT II activity at high body temperature, although this reduction is minimal or mild at normal body temperature. The thermal instability and short T1/2 of CPT II might play important roles in reducing ATP values below the phenotypic threshold in brain endothelial cells of patients with virus-induced encephalitis, particularly those with hyperpyrexia.18 Under heat stress, fasting, acidosis and seizures, moderately lowered CPT II
Blood ATP Levels in Patients With Acute Encephalitis ATP levels in the extracts of whole blood of patients with CPT II variants in the acute phase of encephalitis during high fever were significantly lower (0.63 ± 0.21 mmol/L, n = 200) compared with those in the convalescent phase (1.21 ± 0.32 mmol/L, n =200, P = 0.003) and those of patients without CPT II variants (0.95 ± 0.27 mmol/L, n =150, P = 0.009).
DISCUSSION CPT II appears to be the product of a single gene that is expressed uniformly in every tissue examined thus far.17 More than 25 different mutations and 3 polymorphisms have been identified in the CPT II gene.11,14,18–23 Most mutations are missense mutations and found in exons 1, 3, 4 and 5. Incidence of acute encephalopathy is higher in East Asians than in Caucasians, suggesting that genetic factors related to metabolism are important etiologic factors. Chen et al9 first revealed a thermolabile phenotype of compound heterozygotes for [1055T>G (p. Phe352Cys)] and [1102G>A (p.Val368Ile)], which shows a higher frequency in IAE patients than healthy volunteers. Next, Yao et al18 found 3 compound CPT II variations in patients with severe encephalopathy, including [1055T>G (p.Phe352Cys); 1102G>A (p.Val368Ile)], [1511C>T (p.Pro504Leu); 1813G>C (p.Val605Leu)] and [1055T>G (p.Phe352Cys); 1102G>A (p.Val368Ile); 1813G>C (p.Val605Leu)], which could cause reduced activities, thermal instability and short half-lives of CPT II compared with its wild type (WT). Clinical and neuropathologic studies have revealed that pathogenesis of IAE is correlated with damage of the BBB and hypercytokinemia.24–27 Because brain capillary endothelium is characterized by a greater density of mitochondria compared with peripheral capillaries and fatty acid oxidation is a major energy source of ATP generation, particularly in brain endothelial cells,
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FIGURE 1. Lymphocyte CPT II activity of patients with rs2229291 and rs1799821. At 41°C, CPT II activity of a patient with rs2229291 decreased to about 60% of that at 35°C and 37°C after 2 hours of incubation (upper). CPT II activity of a patient with rs1799821 was not reduced after 2 hours incubation at 41°C compared with that at 35°C (lower). © 2014 Lippincott Williams & Wilkins
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activity because of the CPT II variant may accelerate the disease process of acute encephalitis. Furthermore, pathogen-induced cytokine storm stimulated virus-cytokine-protease cycle in the endothelial cells and various organs28–30 and this might augment “impaired energy metabolism” and vascular hyperpermeability in endothelial cells with the CPT II variants of encephalitis. In the present study, a larger sample of 440 acute encephalitis patients and 229 healthy children with same age was enrolled. DNA examining results showed that there were significant differences of rs2229291 and rs1799821 in CPT II gene between patients and controls. Frequency of rs2229291 in patients was higher compared with controls, whereas it was lower for rs1799821. This hinted that the 2 polymorphisms might be associated with the development of acute encephalitis. Moreover, there was no statistical difference in compound heterozygotes rs2229291 and rs1799821 between patients and controls. This was not consistent with previous reports, a larger sample might be 1 of the reasons. As Chen et al9 and Kubota et al13 reported, rs2229291 (1055T>G, p. Phe352Cys), which can cause visible thermal instability of CPT II activities of lymphocyte in patients with the Phe352Cys CPT II variant, that is, a marked activity reduction (72% compared with WT CPT II) at 41°C rs1799821 (1102G>A, p. Val368Ile), which cause a slight activity reduction (91% compared with WT CPT II) at 41°C. Also, our further expression and function investigations of CPT II might help to elucidate the mechanisms. We further examined the CPT II activity of patients with CPT II variants at different temperature and found CPT II activity in these patients decreased at high temperature (39°C and 41°C) compared with normal temperature (35°C and 37 °C). Gene variants of CPT II impaired its function and resulted in secondary CPT II deficiency, this was consistent with Kubota’s report.13 CPT II activity of patients with rs2229291 decreased obviously at high temperature whereas it was not for patients with rs1799821, it is very interesting to make clear the role of the 2 gene variants on CPT II deficiency. In Yao et al11 study, lysates of COS-7 cells expressing these CPT II mutants were preincubated under high temperature of 41°C for 2 hours as well as under stable condition at 30°C, and then CPT II activities were measured at 30°C. Mild thermal instability of CPT II Phe352Cys and Gln216Arg was detected at 41°C and the instability was markedly enhanced at 41°C by the additional compound mutations of Val368Ile and Met647Val, although that of WT was well-maintained at above 90% under the same conditions. So it is not a general heat denaturation for high body temperature to inactivate CPT II protein. Yao et al18 have shown that COS-7 cells transfected with thermolabile (Phe352Cys + Val368Ile) CPT II variants exhibited significantly decreased fatty acid oxidation and subsequent intracellular ATP reduction at 41°C. In our study, blood ATP concentrations in the acute phase of encephalitis of patients with CPT II variants during high fever were significantly lower than those in the convalescent phase and those without CPT II variants. This suggested that mitochondrial energetic failure may be more severe in patients with CPT II variants. BBB is particularly susceptible to acute hypoxic insult under the inadequate supply of ATP. From a similar perspective, BBB breakdown may occur at an initial stage of encephalitis under the condition of ATP reduction, thus leading to subsequent brain edema because of complex cascade of hypercytokinemia, excitotoxicity and oxidative stress. Although there is 1 pathomechanism that cytokine storm because of virus-glial cell interaction might cause endothelial cell damage (BBB breakdown) leading to brain edema and neuronal injury, we consider that endothelial cell damage might induce in turn cytokine production resulting in neuronal damage in patients with CPT II variant. © 2014 Lippincott Williams & Wilkins
CPT II Gene and Encephalitis
The present study has some limitations. First, we did not examine the expression of CPT II gene with these variants carefully to investigate the effect and mechanism of the variants on expression and function of CPT II protein. These are what we will do in our further investigations. Second, more predisposing factors of the development of acute encephalitis, such as other variants in CPT II gene or other genes, need to be discovered. In the present study, frequencies of rs2229291 and rs1799821 variants in CPT II gene were statistically different between patients and controls; CPT II activity and blood ATP levels of patients with these variants showed homologous change. rs2229291 and rs1799821 variants in CPT II gene might be 1 of the predisposing factors of acute encephalitis. However, the predisposing factors and mechanisms of the development of acute encephalitis need further investigations.
ACKNOWLEDGMENTS We appreciate all subjects for participating in this study. REFERENCES 1. Hsiao YS, Jogl G, Esser V, et al. Crystal structure of rat carnitine palmitoyltransferase II (CPT-II). Biochem Biophys Res Commun. 2006;346:974–980. 2. Joshi PR, Deschauer M, Zierz S. Clinically symptomatic heterozygous carnitine palmitoyltransferase II (CPT II) deficiency. Wien Klin Wochenschr. 2012;124:851–854. 3. Wataya K, Akanuma J, Cavadini P, et al. Two CPT2 mutations in three Japanese patients with carnitine palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes. Hum Mutat. 1998;11:377–386. 4. Yasuno T, Kaneoka H, Tokuyasu T, et al. Mutations of carnitine palmitoyltransferase II (CPT II) in Japanese patients with CPT II deficiency. Clin Genet. 2008;73:496–501. 5. Tadokoro R, Okumura A, Nakazawa T, et al. Acute encephalopathy with biphasic seizures and late reduced diffusion associated with hemophagocytic syndrome. Brain Dev. 2010;32:477–481. 6. Takanashi J, Oba H, Barkovich AJ, et al. Diffusion MRI abnormalities after prolonged febrile seizures with encephalopathy. Neurology. 2006;66:1304– 9; discussion 1291. 7. Ichiyama T, Suenaga N, Kajimoto M, et al. Serum and CSF levels of cytokines in acute encephalopathy following prolonged febrile seizures. Brain Dev. 2008;30:47–52. 8. Matsumoto H, Hatanaka D, Ogura Y, et al. Severe human herpesvirus 6-associated encephalopathy in three children: analysis of cytokine profiles and the carnitine palmitoyltransferase 2 gene. Pediatr Infect Dis J. 2011;30:999–1001. 9. Chen Y, Mizuguchi H, Yao D, et al. Thermolabile phenotype of carnitine palmitoyltransferase II variations as a predisposing factor for influenzaassociated encephalopathy. FEBS Lett. 2005;579:2040–2044. 10. Shinohara M, Saitoh M, Takanashi J, et al. Carnitine palmitoyl transferase II polymorphism is associated with multiple syndromes of acute encephalopathy with various infectious diseases. Brain Dev. 2011;33:512–517. 11. Yao D, Yao M, Yamaguchi M, et al. Characterization of compound missense mutation and deletion of carnitine palmitoyltransferase II in a patient with adenovirus-associated encephalopathy. J Med Invest. 2011;58:210–218. 12. Yao M, Yao D, Yamaguchi M, et al. Bezafibrate upregulates carnitine palmitoyltransferase II expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza-associated encephalopathy. Mol Genet Metab. 2011;104:265–272. 13. Kubota M, Chida J, Hoshino H, et al. Thermolabile CPT II variants and low blood ATP levels are closely related to severity of acute encephalopathy in Japanese children. Brain Dev. 2012;34:20–27. 14. Mak CM, Lam CW, Fong NC, et al. Fatal viral infection-associated encephalopathy in two Chinese boys: a genetically determined risk factor of thermolabile carnitine palmitoyltransferase II variants. J Hum Genet. 2011;56:617–621. 15. Chida J, Yao D, Wang S, et al. [Description of the mechanism of influenza encephalopathy development]. Jpn J Antibiot. 2009;62 (suppl A):74–77. 16. Shiomi M. [Pathogenesis of acute encephalitis and acute encephalopathy]. Nihon Rinsho. 2011;69:399–408.
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17. Rufer AC, Thoma R, Benz J, et al. The crystal structure of carnitine palmitoyltransferase 2 and implications for diabetes treatment. Structure. 2006;14:713–723. 18. Yao D, Mizuguchi H, Yamaguchi M, et al. Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy. Hum Mutat. 2008;29:718–727. 19. Kido H, Yao D, Le Trong Q, et al. [Analysis of SNPs and enzymatic disorder in the patients of influenza-associated encephalopathy: disorder of fatty acid metabolism in mitochondria induced by high fever]. Nihon Rinsho. 2006;64:1879–1886. 20. Olpin SE, Afifi A, Clark S, et al. Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency. J Inherit Metab Dis. 2003;26:543–557. 21. Aoki J, Yasuno T, Sugie H, et al. A Japanese adult form of CPT II deficiency associated with a homozygous F383Y mutation. Neurology. 2007;69:804–806. 22. Isackson PJ, Sutton KA, Hostetler KY, et al. Novel mutations in the gene encoding very long-chain acyl-CoA dehydrogenase identified in patients with partial carnitine palmitoyltransferase II deficiency. Muscle Nerve. 2013;47:224–229. 23. Isackson PJ, Bennett MJ, Vladutiu GD. Identification of 16 new diseasecausing mutations in the CPT2 gene resulting in carnitine palmitoyltransferase II deficiency. Mol Genet Metab. 2006;89:323–331.
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24. Kakita H, Aoyama M, Nagaya Y, et al. Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production. Toxicol Appl Pharmacol. 2013;268:99–105. 25. Asai H, Kakita H, Aoyama M, et al. Diclofenac enhances proinflammatory cytokine-induced aquaporin-4 expression in cultured astrocyte. Cell Mol Neurobiol. 2013;33:393–400. 26. Morichi S, Kawashima H, Ioi H, et al. High production of interleukin-10 and interferon-γ in influenza-associated MERS in the early phase. Pediatr Int. 2012;54:536–538. 27. Hasegawa S, Matsushige T, Inoue H, et al. Serum and cerebrospinal fluid cytokine profile of patients with 2009 pandemic H1N1 influenza virus-associated encephalopathy. Cytokine. 2011;54:167–172. 28. Mizuguchi M, Yamanouchi H, Ichiyama T, et al. Acute encephalopathy associated with influenza and other viral infections. Acta Neurol Scand. 2007;115(4 suppl):45–56. 29. Fukumoto Y, Okumura A, Hayakawa F, et al. Serum levels of cytokines and EEG findings in children with influenza associated with mild neurological complications. Brain Dev. 2007;29:425–430. 30. Wang S, Le TQ, Kurihara N, et al. Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza. J Infect Dis. 2010;202:991–1001.
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Association of CPT II Gene With Risk of Acute Encephalitis in Chinese Children Jianxia Hu, MD,* Zongbo Chen, MD,† Xiaoyi Liu, MD,‡ Zhihong Chen, MD,† Dandan Xin, MD,† and Peipei Liu, MD† Background: Mutations of the CPT II gene cause CPT II deficiency, an inborn metabolic error affecting mitochondrial fatty acid β-oxidation. Associations and mechanisms of CPT II gene with acute encephalitis need to be elucidated. We aimed to investigate the associations of CPT II gene variants and CPT II activity with development of acute encephalitis. Methods: A total of 440 blood-unrelated Chinese children with acute encephalitis and 229 healthy controls were enrolled in this case control study. Sequencing of 5 exons of the CPT II gene was carried out to look for the variants associated with acute encephalitis. CPT II activity and blood adenosine triphosphate concentration were examined during high fever and convalescent phase to confirm the hypothesis. Results: Polymorphism of rs2229291 in CPT II gene was significantly associated with an increased risk of acute encephalitis (P = 0.031), where as rs1799821 displayed a decrease risk (P = 0.018). Positive association was found between rs2229291 and patients with fever at onset of seizure and degree of pathogenetic condition (P = 0.018 and P = 0.023), but not for rs1799821. CPT II activity of patients with rs2229291 reduced greatly during high fever compared with the convalescent phase. Conclusions: rs2229291 and rs1799821 variants in CPT II gene might be 1 of the predisposing factors of acute encephalitis. Key Words: carnitine palmitoyl transferase II, acute encephalitis, polymorphisms (Pediatr Infect Dis J 2014;33:1077–1082)
childhood in East Asians. Chen et al9 discovered a significant portion of patients who had severe influenza-associated encephalopathy (IAE) and showed abnormal acylcarnitine patterns indicating a failing mitochondrial fatty acid oxidation. Shinohara et al10 confirmed these findings in 29 Japanese patients with acute encephalopathy. Also, some reports have presumed that genotypic variants of CPT II gene are associated with fatal or severe cases of IAE.11–14 Mutations of the CPT II gene cause CPT II deficiency, an inborn metabolic error affecting mitochondrial fatty acid β-oxidation. When patients with CPT II deficiency are infected with viruses, some develop energy failure and show a clinical course resembling that of acute encephalopathy.15 There is a great density of mitochondria to supply constant adenosine triphosphate (ATP) in the brain capillary endothelium which can maintain the physiologic functions of significant active transport mechanisms, electrochemical gradients, autoregulatory adjustments and regulation of tight junctional complexes. CPT II deficiency might cause lower blood ATP values in the acute phase of encephalopathy during high fever. Thus the blood brain barrier (BBB) breakdown may occur at an initial stage of encephalopathy under the condition of ATP reduction, thus leading to subsequent brain edema.16 However, there are few reports about the mechanisms and associations between CPT II gene variants and acute encephalitis. In the present study, we investigated the associations and possible mechanisms of CPT II gene variants with acute encephalitis.
MATERIALS AND METHODS
C
arnitine palmitoyl transferase II (CPT II) is a catalytically active, malonyl CoA insensitive enzyme localized on the mitochondrial inner membrane and plays a key role in the transport of long-chain fatty acids into the mitochondria. CPT II facilitates the β-oxidation of long-chain fatty acids in the mitochondria and is ubiquitously expressed in all tissues that require fatty acid oxidation as an energy-producing pathway.1,2 CPT II deficiency can induce a disorder of long-chain fatty acids oxidation and impaired energy production, the effects of which are most pronounced during fasting or infection, when fatty acid breakdown is an essential energy source.3,4 Acute encephalitis is an acute brain dysfunction that usually occurs at the early stage of infectious diseases with symptoms of high fever, impaired consciousness, convulsions or seizures associated with brain edema.5,6 The antecedent infection of acute encephalitis is usually viral, such as human enterovirus, human herpes virus-6 and some other viruses,7–9 and its incidence is highest in infancy and early Accepted for publication March 22, 2014. From the *Stem Cell Center; †Department of Paediatrics; and ‡Department of Galactophore, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, China. The authors have no funding or conflicts of interest to disclose. Address for correspondence: Zongbo Chen, Department of Paediatrics, the Affiliated Hospital of Medical College, Qingdao University, No. 59, Haier Road, Qingdao 266000, China. E-mail: [email protected]. Copyright © 2014 by Lippincott Williams & Wilkins ISSN: 0891-3668/14/3310-1077 DOI: 10.1097/INF.0000000000000368
Patients The study protocol was approved by the Ethical Committee of the Affiliated Hospital of the Medical College, Qingdao University. Informed consent according to the Declaration of Helsinki was provided by every patient’s guardian. Between May 2010 and November 2012, 600 patients were screened for enrollment in the Department of Pediatrics of our hospital. Of these patients, 82 patients were excluded for diagnosis as bacterial encephalitis, 518 patients fitted the inclusion criteria and were personally interviewed. After that, 440 patients with acute encephalitis were enrolled whereas other 78 patients refused to participate in the study. All patients were diagnosed as viral encephalitis according to the clinical manifestations and laboratory examinations of the disease. Patients with the abrupt onset of febrile convulsive status epilepticus and cataphora were considered as severe degree of pathogenetic condition. Patients with the abrupt onset of febrile convulsive status epilepticus but not with consciousness disorder were considered as mild degree of pathogenetic condition. Viral nucleic acids from blood, feces and cerebrospinal fluid were examined by reverse transcription-polymerase chain reaction (PCR). Patients with other metabolism disorders, cardiopathy, dysgnosia, hypoplasia or cerebral palsy caused by innateness, trauma or other nonviral infections were excluded.
Controls Two-hundred and twenty-nine healthy children volunteers with a similar history of virus infection but without history of encephalitis, including 112 male and 117 female, at 2–8 years of
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age, were enrolled as controls. The conventional treatment for controls was rest, drinking more water, vitamin supplement and antiviral therapy (eg, Rimantadine). Volunteers with other metabolism disorders, cardiopathy, dysgnosia, congenital hypoplasia or cerebral palsy, trauma or other nonviral infections were excluded. All their previous histories were carefully recorded.
Clinical Data Analysis For all subjects, patients and controls, during the treatment, the hyperthermia, duration of fever, blood routine, electroencephalogram, cerebrospinal fluid routine, computerized tomography or magnetic resonance imaging (MRI) of brain and electrocardiogram were all examined and carefully recorded. All symptoms including headache, nausea, vomit, lethargy, clouding of consciousness, cataphora, convulsion, meningeal irritation sign, ataxia, respiratory or circulatory failure, abdominal pain, diarrhea, rash, arthralgia and pharyngalgia were recorded carefully.
Genomic DNA Extraction Genomic DNA was extracted from peripheral blood using a commercial blood Genomic DNA extraction kit (from Dongsheng Biotech, Beijing, China) according to the manufacturer’s instructions. The extracted DNA samples were stored in a freezer at −80°C until genetic polymorphism analyses were performed.
Genotyping Genomic DNA from whole blood was evaluated by electrophoresis (1% agarose) and diluted to a working concentration of 5–10 ng/µL. PCR of 5 exons of the CPT II gene was carried out with intron-based primers in genomic DNA. For P2F1/R1 segment of CPT II gene, the reaction condition was as following: a 10 µL mixture was prepared for each reaction and included 1× GC buffer, 0.2 mmol/L dNTP, 0.2 µmol/L of each primer, 0.25 units Ex Taq polymerase (Takara, Dalian, China) and 1 µL template DNA. The cycling program was 95°C for 5 minutes; 35 cycles of 96°C for 10 seconds, 68°C for 1 minute per cycle and 72°C for 2 minutes. For other segments, the reaction condition was as following: a 10 µL mixture was prepared for each reaction and included 1× Ex Taq buffer, 2.0 mmol/L Mg2+, 0.2 mmol/L dNTP, 0.2 µmol/L of each primer, 0.25 units Ex Taq polymerase and 1 µL template DNA. The cycling program was 95°C for 5 minutes; 11 cycles of 94°C for 15 seconds, 62°C for 40 seconds per cycle, 72°C for 1 minute; 24 cycles of 94°C for 15 seconds, 56°C for 30 seconds, 72°C for 1 minute and 72°C for 2 minutes. The sequences of the PCR products were analyzed with the ABI BigDye 3.1 Sequencing Kit on an ABI 3130 XL Genetic Analyzer (Applied Biosystems Inc, Foster City, CA). Reaction mixture included 2 µL BigDye3.1 mix, 2 µL sequencing primer (0.4 µmol/L) and 1–2 µL purified PCR product. The cycling program was 96°C for 1 minute; 28 cycles of 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes. Each PCR product was sequenced in both strands, and all analyses were performed at least twice independently.
Preparation of Subjects’ Lymphoblasts and Culture Blood samples (2 mL) were obtained from subjects, patients and controls, by venipuncture into a sterile EDTA blood collection tube. Lymphocytes were separated from peripheral blood, diluted (1:1, v/v) with sterile saline, by centrifugation (800 g, 20 minutes) over 2 mL of Lymphoprep (Nycomed, Denmark). The lymphocyte layer was recovered and washed twice with phosphate-buffered saline by centrifugation at 250 g for 10 minutes each, and then maintained in PRMI-1640 (GIBCO) supplemented with 12.5% fetal calf serum. Cells were incubated with 5% CO2 at 37°C for
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7 days. Lymphoblastic cell lines were established by infecting peripheral blood lymphocytes with the Epstein Barr virus. Cells were grown in suspension in an SC flask (Greiner 658190, Germany) in an upright position, in 10 mL of PRMI-1640 medium that contained 12.5% fetal calf serum, maintained at 37°C. Fluid was routinely changed every 2 days by removing the medium above the settled cells and replacing it with an equal volume of fresh medium.
Analysis of CPT II Activity CPT II activities of subjects’ lymphoblasts were analyzed as previously described.13 To prepare whole cell extracts, cells were harvested and washed twice with phosphate-buffered saline at 250 g for 10 minutes and then lysed with 0.5 mL of ice-cold lysis buffer (5 mmol/L Tris-HCl buffer, pH 7.4, containing 1% Tween-20 and 0.5 mol/L KCl), then centrifuged at 147,600 g for 1 hour at 4°C. CPT II activities in the lysates of lymphoblasts were measured at 30°C for 2 hours, by detection of the palmitoyl-L-[methyl-3 H] carnitine formed from 200 mmol/L L-[methyl-3 H] carnitine and 50 mmol/L palmitoyl-CoA. The formed palmitoyl-L-[methyl-3H] carnitine was extracted with 1-butanol and radioactivity was counted by a liquid scintillation counter (Model LS 6500; Beckman, Fullerton). To analyze the heat stability of CPT II, cell lysates were preincubated at 35, 37, 39 and 41°C for 0–120 minutes, and enzyme activities were measured by addition of 200 mmol/L L-[methyl-3H] carnitine followed by incubation at 30°C for 2 hours. Protein concentrations in the cell lysates were measured using the BCATM Protein Assay Kit (Thermo SCIENTIFIC, Pittsburgh, PA).
Measurement of Blood ATP Concentrations ATP concentrations in whole blood lysates were measured by an ENLITEN ATP assay system bioluminescence detection kit (Promega, Madison, WI) according to the instructions provided by the manufacturer and the values were expressed as ATP levels in whole blood. Briefly, 0.5% trichloroacetic acid was added in blood for ATP efficient release. Then, 25 mmol/L Tris-acetate (pH 7.75) was used for neutralization. After addition of recombinant luciferase/luciferin reagent (rL/L), luminescence was measured using a 10-second integration time with a microplate luminometer (Lmax) and SOFTmax PRO software (Molecular Devices) and was normalized to protein concentration. The ATP standard curve was generated using the ATP standard (10−7 mol/L) included in the kit.
Statistical Analysis All statistical analyses were conducted using Prism 5. The observed genotype frequencies in controls were tested for the Hardy-Weinberg equilibrium using the χ2 test. Associations between gene variants and risk of acute encephalitis were tested using the χ2 test and odds ratios (OR) and 95% confidence intervals (95% CI) were calculated. Normally distributed, continuous measures are presented as mean ± SD. The differences of CPT II activities and blood ATP levels between high fever and convalescent phase were tested by independent samples t test and paired samples t test. We initially considered all P values < 0.05 as nominally significant but further assessed the significance using the conservative Bonferroni correction to account for multiple comparisons. All P values were subjected to Bonferroni correction for multiple testing by multiplying uncorrected P values by the number of independent tests performed (8 for single nucleotide polymorphism testing). We presented the corrected P values. A difference with a corrected value of P < 0.05 was considered significant.
RESULTS A total of 629 subjects were enrolled in this study, including 440 acute encephalitis patients and 229 controls. All these subjects © 2014 Lippincott Williams & Wilkins
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TABLE 1. Clinical Summary of All Subjects Patients (n = 440)
Variable Age (year) Age below 3 years [n (%)] Gender [n (%)] Girl Boy Pathogen Diagnosis Duration of high fever (day) Hyperthermia (°C) Duration of seizure (min) Degree of pathogenetic condition [n (%)] Mild Severe Electroencephalogram (abnormal) [n (%)] Brain MRI (abnormal) [n (%)] Prognosis [n (%)] Good Poor
Controls (n = 229)
3.9 ± 1.8 303 (68.9%)
4.6 ± 2.0 163 (71.2%)
227 (51.6%) 213 (48.4%) Virus Acute encephalitis 2.3 ± 1.9 38.3 ± 3.1 15.6 ± 10.2
117 (51.1%) 112 (48.9%)
365 (83.0%) 75 (17.0%) 362 (82.3%) 289 (65.7%) 399 (90.7%) 41 (9.3%)
Data of age, duration of high fever, hyperthermia, and duration of seizure are presented as mean ± SD.
are Chinese Han people. Clinical parameters of all subjects are shown in Table 1. About 70% of patients were 0.99). Table 2 showed the risks of acute encephalitis in relation to the 2 CPT II variants. The proportions of the TT, TG and GG genotypes of rs2229291 in controls and patients were 65.1%, 28.4% and 6.5% and 54.5%, 37.7% and 7.8%, respectively, whereas, for rs1799821, the proportions of the AA, AG and GG genotypes in controls and patients were 47.6%, 41.0% and 11.4% and 55.0%, 39.3% and 5.7%, respectively. There were statistical differences in the 2 polymorphisms between patients and controls (P = 0.031 and P = 0.018). As shown in Table 2, the TG+GG genotypes of rs2229291 were markedly overrepresented in the patients compared with controls (P = 0.009 with Bonferroni correction), whereas GG genotype of rs1799821 was significantly lower in patients compared with controls (P = 0.005 with Bonferroni correction). By respectively using the TT and AA genotype as reference, significant associations were found between the TG (rs2229291) and GG (rs1799821) genotypes and the risk of acute encephalitis (OR = 1.346 and OR = 2.309, respectively; P = 0.010 and P = 0.005 with Bonferroni correction). After Bonferroni adjustment, there were statistically significant associations between rs1799821 G allele (P = 0.011, OR = 1.379, 95% CI: 1.075–1.767) and acute encephalitis. Furthermore, no statistical differences in compound heterozygotes of rs2229291 and rs1799821 were found between patients and controls (P = 0.697).
Associations of rs2229291 and rs1799821 With Clinical Characteristics To investigate whether the genotypes relate to different clinical characteristics, including temperature at onset of seizure (≤39°C or >39°C), degree of pathogenetic condition (mild or severe), electroencephalogram (normal or abnormal), brain MRI (normal or abnormal) and prognosis (good or poor), we compared genotype distributions between patients with these clinical characteristics. Significant differences were found in the TG+GG genotypic frequencies of rs2229291 between patients with temperature at onset of seizure (≤ 39°C), degree of pathogenetic condition (severe; P =0.018 and P=0.023 with Bonferroni correction, showed in Table 3). No significant associations were found between rs1799821 and clinical characteristics.
TABLE 2. The Genotype and Allele Distributions of rs2229291 and rs1799821 in CPT II Gene Between Patients and Controls Polymorphism rs2229291 Genotypes TT TG GG TG+GG Alleles T G rs1799821 Genotypes AA AG GG AG + GG Alleles A G
Patients [n(%)]
Controls [n(%)]
χ2
OR
95% CI
240 (54.5%) 166 (37.7%) 34 (7.8%) 200 (45.5%)
149 (65.1%) 65 (28.4%) 15 (6.5%) 80 (34.9%)
0.010 0.294 0.009
1.346 0.711 1.301
1.065–1.701 0.374–1.349 1.061–1.596
646 (73.4%) 234 (26.6%)
363 (79.3%) 95 (20.7%)
0.018
1.246
1.032–1.504
242 (55.0%) 173 (39.3%) 25 (5.7%) 198 (45.0%)
109 (47.6%) 94 (41.0%) 26 (11.4%) 120 (52.4%)
0.276 0.005 0.069
0.882 2.309 0.823
0.704–1.105 1.275–4.181 0.667–1.016
657 (74.7%) 223 (25.3%)
312 (68.1%) 146 (31.9%)
0.011
1.379
1.075–1.767
Tests of independence between gene variants and risk of acute encephalitis were tested using the χ2 test. OR and 95% CI provide the association between gene variants and WT and group (acute encephalitis vs. controls).
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TABLE 3. The Association Between rs2229291 and Clinical Characteristics of Patients Parameters
TT [n (%)]
Temperature at onset of seizure ≤39°C 93 (48.2%) >39°C 147 (59.5%) Degree of pathogenetic condition Mild 208 (57.0%) Severe 32 (42.7%) Electroencephalogram Normal 42 (53.8%) Abnormal 198(54.7%) Brain MRI Normal 92 (60.9%) Abnormal 148 (51.2%) Prognosis Good 215 (53.9%) Poor 25 (61.0%)
TG + GG [n(%)]
χ2
OR
95% CI
100 (51.8%) 100 (40.5%)
0.018
1.280
1.044–1.568
157 (43.0%) 43 (57.3%)
0.023
1.780
1.077–2.942
36 (46.2%) 164 (45.3%)
0.891
0.966
0.591–1.579
59 (39.1%) 141 (48.8%)
0.052
0.801
0.635–1.010
184 (46.1%) 16 (39.0%)
0.385
0.748
0.387–1.443
Tests of independence between gene variants and clinical characteristics of acute encephalitis were tested using the χ2 test. OR and 95% CI provide the association between gene variants and clinical characteristics.
Lymphocyte CPT II Activity in the Patients All patients with rs2229291 variant (n = 200) showed significant reductions of CPT II activity at 41°C (average slip 35 ± 18.3%, P = 0.001). As shown in Fig. 1, CPT II activity using peripheral lymphocytes of a patient with rs2229291 was significantly reduced to about 60% during incubation for 120 minutes at 41°C compared with those at 35°C and 37°C. CPT II activities in patients with rs1799821 variant (n = 198) were not reduced obviously during incubation for 120 min at 41°C compared with that at 35°C (average slip 10 ± 5.9%, P = 0.13).
the CPT II variants may cause mitochondrial fuel usage failure during high fever in the brain endothelial cells. Mutations of the CPT II gene cannot cause low rates of protein synthesis but can cause a severe reduction of CPT II activity at high body temperature, although this reduction is minimal or mild at normal body temperature. The thermal instability and short T1/2 of CPT II might play important roles in reducing ATP values below the phenotypic threshold in brain endothelial cells of patients with virus-induced encephalitis, particularly those with hyperpyrexia.18 Under heat stress, fasting, acidosis and seizures, moderately lowered CPT II
Blood ATP Levels in Patients With Acute Encephalitis ATP levels in the extracts of whole blood of patients with CPT II variants in the acute phase of encephalitis during high fever were significantly lower (0.63 ± 0.21 mmol/L, n = 200) compared with those in the convalescent phase (1.21 ± 0.32 mmol/L, n =200, P = 0.003) and those of patients without CPT II variants (0.95 ± 0.27 mmol/L, n =150, P = 0.009).
DISCUSSION CPT II appears to be the product of a single gene that is expressed uniformly in every tissue examined thus far.17 More than 25 different mutations and 3 polymorphisms have been identified in the CPT II gene.11,14,18–23 Most mutations are missense mutations and found in exons 1, 3, 4 and 5. Incidence of acute encephalopathy is higher in East Asians than in Caucasians, suggesting that genetic factors related to metabolism are important etiologic factors. Chen et al9 first revealed a thermolabile phenotype of compound heterozygotes for [1055T>G (p. Phe352Cys)] and [1102G>A (p.Val368Ile)], which shows a higher frequency in IAE patients than healthy volunteers. Next, Yao et al18 found 3 compound CPT II variations in patients with severe encephalopathy, including [1055T>G (p.Phe352Cys); 1102G>A (p.Val368Ile)], [1511C>T (p.Pro504Leu); 1813G>C (p.Val605Leu)] and [1055T>G (p.Phe352Cys); 1102G>A (p.Val368Ile); 1813G>C (p.Val605Leu)], which could cause reduced activities, thermal instability and short half-lives of CPT II compared with its wild type (WT). Clinical and neuropathologic studies have revealed that pathogenesis of IAE is correlated with damage of the BBB and hypercytokinemia.24–27 Because brain capillary endothelium is characterized by a greater density of mitochondria compared with peripheral capillaries and fatty acid oxidation is a major energy source of ATP generation, particularly in brain endothelial cells,
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FIGURE 1. Lymphocyte CPT II activity of patients with rs2229291 and rs1799821. At 41°C, CPT II activity of a patient with rs2229291 decreased to about 60% of that at 35°C and 37°C after 2 hours of incubation (upper). CPT II activity of a patient with rs1799821 was not reduced after 2 hours incubation at 41°C compared with that at 35°C (lower). © 2014 Lippincott Williams & Wilkins
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activity because of the CPT II variant may accelerate the disease process of acute encephalitis. Furthermore, pathogen-induced cytokine storm stimulated virus-cytokine-protease cycle in the endothelial cells and various organs28–30 and this might augment “impaired energy metabolism” and vascular hyperpermeability in endothelial cells with the CPT II variants of encephalitis. In the present study, a larger sample of 440 acute encephalitis patients and 229 healthy children with same age was enrolled. DNA examining results showed that there were significant differences of rs2229291 and rs1799821 in CPT II gene between patients and controls. Frequency of rs2229291 in patients was higher compared with controls, whereas it was lower for rs1799821. This hinted that the 2 polymorphisms might be associated with the development of acute encephalitis. Moreover, there was no statistical difference in compound heterozygotes rs2229291 and rs1799821 between patients and controls. This was not consistent with previous reports, a larger sample might be 1 of the reasons. As Chen et al9 and Kubota et al13 reported, rs2229291 (1055T>G, p. Phe352Cys), which can cause visible thermal instability of CPT II activities of lymphocyte in patients with the Phe352Cys CPT II variant, that is, a marked activity reduction (72% compared with WT CPT II) at 41°C rs1799821 (1102G>A, p. Val368Ile), which cause a slight activity reduction (91% compared with WT CPT II) at 41°C. Also, our further expression and function investigations of CPT II might help to elucidate the mechanisms. We further examined the CPT II activity of patients with CPT II variants at different temperature and found CPT II activity in these patients decreased at high temperature (39°C and 41°C) compared with normal temperature (35°C and 37 °C). Gene variants of CPT II impaired its function and resulted in secondary CPT II deficiency, this was consistent with Kubota’s report.13 CPT II activity of patients with rs2229291 decreased obviously at high temperature whereas it was not for patients with rs1799821, it is very interesting to make clear the role of the 2 gene variants on CPT II deficiency. In Yao et al11 study, lysates of COS-7 cells expressing these CPT II mutants were preincubated under high temperature of 41°C for 2 hours as well as under stable condition at 30°C, and then CPT II activities were measured at 30°C. Mild thermal instability of CPT II Phe352Cys and Gln216Arg was detected at 41°C and the instability was markedly enhanced at 41°C by the additional compound mutations of Val368Ile and Met647Val, although that of WT was well-maintained at above 90% under the same conditions. So it is not a general heat denaturation for high body temperature to inactivate CPT II protein. Yao et al18 have shown that COS-7 cells transfected with thermolabile (Phe352Cys + Val368Ile) CPT II variants exhibited significantly decreased fatty acid oxidation and subsequent intracellular ATP reduction at 41°C. In our study, blood ATP concentrations in the acute phase of encephalitis of patients with CPT II variants during high fever were significantly lower than those in the convalescent phase and those without CPT II variants. This suggested that mitochondrial energetic failure may be more severe in patients with CPT II variants. BBB is particularly susceptible to acute hypoxic insult under the inadequate supply of ATP. From a similar perspective, BBB breakdown may occur at an initial stage of encephalitis under the condition of ATP reduction, thus leading to subsequent brain edema because of complex cascade of hypercytokinemia, excitotoxicity and oxidative stress. Although there is 1 pathomechanism that cytokine storm because of virus-glial cell interaction might cause endothelial cell damage (BBB breakdown) leading to brain edema and neuronal injury, we consider that endothelial cell damage might induce in turn cytokine production resulting in neuronal damage in patients with CPT II variant. © 2014 Lippincott Williams & Wilkins
CPT II Gene and Encephalitis
The present study has some limitations. First, we did not examine the expression of CPT II gene with these variants carefully to investigate the effect and mechanism of the variants on expression and function of CPT II protein. These are what we will do in our further investigations. Second, more predisposing factors of the development of acute encephalitis, such as other variants in CPT II gene or other genes, need to be discovered. In the present study, frequencies of rs2229291 and rs1799821 variants in CPT II gene were statistically different between patients and controls; CPT II activity and blood ATP levels of patients with these variants showed homologous change. rs2229291 and rs1799821 variants in CPT II gene might be 1 of the predisposing factors of acute encephalitis. However, the predisposing factors and mechanisms of the development of acute encephalitis need further investigations.
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