INTRODUCTION

Acute encephalopathy is the most serious complication of pediatric viral infections. Acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) is the most frequent type of acute encephalopathy in Japan. However its etiology and pathogenesis remain unclear. Its incidence is highest in infancy and early childhood. The antecedent infections are caused by various viruses and bacteria. However, there is no specific difference between the antecedent infections and the type of acute encephalopathy. In addition, the frequency of acute encephalopathy in East Asians is higher than that in Caucasians. This suggests that genetic factors play an important role in the etiology of acute encephalopathy.

OBJECTS

Acute encephalopathy has been considered multifactorial syndrome, caused by multiple genetic and environmental factors. However, most of these risk factors, especially for genetic factors, have been unknown. In order to develop the effective treatments and the prevention methods for AESD, it is necessary to investigate genetic background in the patients with AESD. It has recently been reported that thermolabile haplotype 352C-368I-647M of the CPT2 (carnitine palmitoyl transferase 2 gene) is associated with influenza encephalopathy with poor prognosis. There is no report conducting CPT2 variations analysis in a large number of AESD patients with various antecedent infections. In addition, I hypothesized that the adenosine-mediated signal pathway may be altered in AESD. Adenosine is an endogenous anticonvulsant. And theophylline, which is non-selective A1R and A(2A)R antagonist, is considered one environmental risk factor of AESD. Dysfunction of adenosine pathway might be caused excitotoxicity and predispose AESD. Therefore, this study focused on three genes, carnitine palmitoyltransferase 2 gene (CPT2), Adenosine A1R and A(2a)R gene (ADORAI and ADORA2A) in AESD patients.

MATERIALS

85 patients with AESD from hospitals in Japan and 100 normal controls from Human science research resources bank participated in genetic analysis. And in order to investigate ADORA2A mRNA expression level, 100 human brain cDNA and RNA samples were obtained from Stanley Medical Research Institute (SMRI) (Bethesda, MD, USA). In order to investigate mRNA and protein expression level and estimate cAMP accumulation, 15 lymphoblast cell lines from control Japanese adults, obtained from control subjects at the University of Tokyo Hospital for functional analysis.

METHODS

After informed consent, genomic DNA of patients was extracted from whole blood using standard protocols. First, three CPT2 SNPs (rs2229291 c.1055T>G p.F352C and rs1799821 c.1102A>G p.I368V in exon4, and rs1799822 c.1939A>G p.M647V in exon5) were analyzed in 85 patients with AESD and 100 controls. TA cloning was performed, when patients and controls showed heterozygosity in two SNPs, rs2229291 and rs1799821, of exon 4. Second, ADORAI and ADORA2A in 85 patients with AESD and 100 normal controls were analyzed. For statistical analysis, goodness-of-fit to the Hardy-Weinberg equilibrium in patients and control subjects were examined by χ2 test using Microsoft Office Excel 2010. Differences in the demographic characteristics of the genotypes were assessed by Pearson's χ2 test and Fisher's exact test for categorical data. Odds ratio (OR) together with the 95% confidence interval (CI) for each allele and haplotype frequency with AESD were calculated by Microsoft Office Excel 2010. Bonferroni correction was used to adjust for multiple testing. Significant differences were defined as a corrected p value in each conditional analysis. Finally, the mRNA expression in brain samples, mRNA and protein expression in lymphoblasts, as well as the production of cyclic adenosine monophosphate (cAMP) by lymphoblasts in response to adenosine was compared among ADORA2A diplotypes. The differences in mRNA and protein expression levels and cellular cAMP accumulation, expressed as the mean ± SEM, were calculated using analysis of variance (ANOVA) followed by the Tukey-Kramer test in the case of multiple comparisons.

RESULTS

By CPT2 analysis, the frequency of rs2229291 G (p.352 C) allele in CPT2 exon 4 was significantly higher in patients than that in controls (p=0.013, OR=2.09, 95%CI=1.22-3.57). 352 C-CPT2 decreases CPT2 activity to 50% of 352 F-CPT2 and is related to the severity of acute encephalopathy. However, the frequency of cases positive rs2229291 G (p352 C) allele did not show difference between good and poor prognosis (p=0.177).

By A1R and A(2A)R analysis, I found ADORA2A AA diplotypes, containing four variations in ADORA2A, was associated with AESD. The frequency of haplotype A (rs2298383 C, rs5751876 T, rs35320474 deletion and rs4822492 C) in patients was significantly higher than in controls (p=0.005, OR=1.70, 95%CI=1.17-2.45). A(2A)R mRNA expression was significantly higher in AA than AB and BB diplotypes, both in the brain (p=0.003 and 0.002, respectively). In lymphoblasts, A(2A)R protein expression (p=0.028), as well as cellular cAMP production (p=0.0006), was significantly higher in AA than BB diplotype.

DISCUSSION

This study found two genetic risk factors. One is thermolabile 352C-CPT2 variation, and another is ADORA2A AA diplotype, consisting of four ADORA2A genetic variants. When cases with thermolabile 352C-CPT2 are provoked by infection and high fever, they may suffer from energy failure and AESD. High A2AR expression level and high cAMP accumulation, caused by ADORA2A AA diplotypes, may predispose children to AESD by altering the intracellular adenosine/cAMP signal cascade. Based on these findings on molecular pathomechanism, further clinical and laboratory studies are needed to find an appropriate biomarker for early diagnosis, and to develop effective treatments and preventive measures for AESD.

本研究は日本人小児けいれん重積型急性脳症(AESD)患者の遺伝的背景を明らかにするため、日本人小児AESD患者85例を対象に、CPT2, ADORA1, ADORA2Aの遺伝子解析を行い、下記の結果を得ている。

1.ミトコンドリア、マトリックス内膜に存在するcarnitine palmitoyl transferase 2酵素は、ミトコンドリアβ酸化に関与している。本研究では、carnitine palmitoyl transferase 2遺伝子(CPT2)の全長塩基配列解析の結果、CPT2のrs2229291 c.1055 G (p.352 C) の頻度が、患者群で有意に高いことを示した。

先行研究でrs2229291 G を有するCPT2の酵素活性は42℃の場合、37℃の場合と比較して、約50%まで低下することが報告されている。このことから、rs2229291 Gを有するを患者では、感染症に伴う高熱を契機にCPT2の酵素活性が低下し、患者はミトコンドリアβ酸化からエネルギーを得ることが難しくなり、深刻なエネルギー危機に陥いると考えられる。これらの結果から、熱不安定CPT2酵素によるエネルギー危機が、AESDの発症機序の一つであることが示された。

2.アデノシンは内因性の神経修飾物質であり、抗けいれん作用を有している。また非選択的アデノシンA1,A(2A)受容体アンタゴニストであるテオフィリンを服用している患者が感染症を契機に、重度のAESDを発症したケースが報告されており、テオフィリンはAESDの環境要因の一つであると考えられている。そこで中枢神経系でアデノシンA1,A2A受容体の、なんらかの機能異常が、AESDの病理学的特徴である興奮毒性を引き起こすのではないかと考え、アデノシンA1,A(2A)受容体遺伝子(ADORA1, ADORA2A)の全長遺伝子解析を行った。結果、ADORA2Aのrs2298383 C, rs5751876 T, rs35320474 del, rs4822492 CからなるハプロタイプA、さらにハプロタイプAのホモ接合ディプロタイプ AAが、患者群で有意に高頻度であることを示した。

3.脳サンプルから抽出したRNAを用いて、Quantitive-Real-Time PCR法により発現量を測定し、ディプロタイプで比較した結果、ADORA2Aのディプロタイプ AAでmRNAの発現量が増加していることを示した。

4.リンパ芽球から抽出したRNA、蛋白を用いて、Quantitive-Real-Time PCR法とWestern blotting法により発現量を測定し、ディプロタイプで比較した結果、ADORA2Aのディプロタイプ AAでmRNA、蛋白ともに発現量が増加していることを示した。

5.アデノシンA(2A)受容体はAdenylcyclaseを活性化して、細胞内cAMP産生量を増加させ、cAMPがProtein Kinase Aに働きかえ、神経細胞興奮を引き起こすことが知られている。本研究でリンパ芽球を用いて、ELISA法により細胞内cAMP産生量を測定したところ、ADORA2Aのディプロタイプ AAで細胞内cAMP産生量が増加していることを示した。これらの結果からADORA2Aのディプロタイプ AAが、アデノシンA(2A)受容体の発現量の増加、細胞内cAMP産生量の増加を引き起こし、神経細胞興奮性を促進させていることが考えられる。この結果からADORA2Aのディプロタイプ AAによる細胞内cAMP産生量の亢進が、AESDの病理学的特徴である興奮毒性の一つの要因であることが示された。

以上、本論文は日本人小児けいれん重積型急性脳症(AESD)患者85例の遺伝子解析を行い、熱感受性のCPT2 rs2229291 Gと、ADORA2Aのディプロタイプ AAが、AESDの遺伝的要因であることを明らかにした。本研究まで、けいれん重積型急性脳症単独での遺伝的要因の報告がなく、本研究の結果は疾患の診断、治療法の確立に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。