吡哆醇依赖性癫痫的临床和遗传学特点及尿液哌啶酸的检测

Clinical and genetic characteristics and detection of urinary pipecolic acid in pyridoxine dependent epilepsy

目的 分析吡哆醇依赖性癫痫（PDE）患儿的临床及遗传学特点，建立尿液哌啶酸的检测方法，分析PDE患儿吡哆醇治疗过程中尿液哌啶酸的浓度。方法 对2012年4月至2015年9月在北京大学第一医院儿科确诊的12例PDE患儿（男8例、女4例）的临床表现、诊治过程、脑电图及神经影像学资料进行回顾性分析；采用Sanger测序或靶向捕获二代测序技术进行ALDH7A1基因检测；采用气相色谱－质谱（GC－MS）方法测定PDE患儿的尿液哌啶酸浓度，并检测非PDE患儿的尿液哌啶酸作为正常对照。对照组为2015年11月至2016年1月于北京大学第一医院出生的新生儿或因晕厥等原因（不影响哌啶酸代谢）就诊的儿童共58例，其中≤6月龄组25例（男14例、女11例），〉6月龄组33例（男14例、女19例）。哌啶酸浓度的组间比较采用两独立样本的t检验或Mann－Whitney检验；相关性分析采用Pearson或Spearman检验。结果 12例PDE患儿中7例母亲妊娠期或出生史异常，起病年龄为生后5 h-5个月，8例于生后10 d内起病。经过15 d-20个月的延迟诊断，患儿的癫痫发作最终均被吡哆醇单药控制，其中10例发作控制时吡哆醇剂量高于10.0 mg/（kg·d），另2例分别为8.5和2.5 mg/（kg·d）。发作间期脑电图显示10例有非特异性异常，2例吡哆醇用药前后均正常；头颅磁共振成像显示7例为非特异性异常，5例正常。患儿均通过ALDH7A1基因检测确诊，共发现15种突变位点，其中4种为国际尚未报道的新突变。6例携带IVS11＋1G〉A突变，出现率25%（6/24）。至末次随访，11例患儿有不同程度的智力、运动发育落后，其中4例重度落后者出生史均有异常；1例未发现明显的发育异常。正常对照组中，≤6月龄组尿液哌啶酸浓度明显高于〉6月龄组[8.47（0.46-35.33）比0.66（0.12-3.52）mmol/mol肌酐，Z＝－5.464，P〈0.01]。12例PDE患儿检测时年龄均〉6月龄，尿液哌啶酸浓度0.14-4.08 mmol/mol肌酐（仅有1例浓度稍高于正常值上限），与正常对照〉6月龄组差异无统计学意义（Z＝－0.655，P〉0.05），与吡哆醇初始、末次随访维持剂量无相关性（r＝0.418、0.166，P＝0.176、0.697）。 结论 PDE患儿多在新生儿早期起病。IVS11＋1G〉A突变出现率较高，可能为我国PDE患儿的热点突变。多数患儿癫痫发作控制后遗留不同程度的智力、运动发育落后，出生史异常者提示预后较差。未发现诊断延迟时间与智力、运动发育情况之间存在相关性。尿液哌啶酸浓度经吡哆醇长期维持治疗后可能恢复正常。

Objective To analyze the clinical and genetic characteristics of patients with pyridoxine dependent epilepsy （PDE） , and build a method to detect and analyze the concentration of urinary pipeeolic acid in PDE patients receiving pyridoxine treatment. Method Twelve patients （8 were male, 4 were female） were diagnosed as PDE in Peking University First Hospital between April 2012 and September 2015. The clinical manifestations, diagnosis and treatment process, video-electroencephalogram, magnetic resonance imaging were retrospectively analyzed. ALDHTA1 gene was detected using Sanger sequencing or targeted next-generation sequencing. The concentration of urinary pipecolic acid in PDE patients was detected with gas chromatography-mass spectrometry （GC-MS） , as well as in some non-PDE children served as normal control. All controls, 58 cases totally, were neonates born in our hospital or children came to our hospital for reasons such as syncope （without disturbing pipecolie acid metabolism） from November 2015 to January 2016. Of them, 25 were ≤6 months old（ 14 were male, 11 were female） , 33 were 〉6 months old （14 were male, 19 were female）. The Student＇s t-test or Mann-Whitney U test was used for comparing the pipecolic acid between the two groups. Correlation analysis was conducted using Pearson or Spearman test. Result Of the 12 patients, seven of them were abnormal at birth. The age of epilepsy onset was from 5 h to 5 months, within 10 d in 8 patients. After a diagnostic delay time of 15 d to 20 months, seizures in all patients were controlled by pyridoxine monotherapy, at a dose of higher than 10.0 mg/（ kg·d） in 10 patients, and 8.5 and 2. 5 mg/（ kg·d） in the other 2 patients respectively. The range of maintenance dose was 2. 5 -20. 0 rag/（ kg·d） during the follow up. Interictal electroencephalogram showed nonspecific abnormality in 10, normal in 2. Brain magnetic resonance imaging showed nonspecific abnormality in 7, normal in 5. ALDH7A1 mutations were found in all patients, including 15 different mutation sites, four of which were never reported before. Splicing mutation IVSll ＋ 1G 〉 A was carried in 6 patients, with a frequency of 25% （6/24）. At the last follow-up, eleven patients were in various degree of psychomotor development delay, including the 4 patients with severe delay in whom birth abnormalities presented, and no significant delay was found in one patient. The concentration of urinary pipecolic acid in control： age ≤ 6 months, median 8.47 （0. 46 - 35.33） mmol/mol creatinine; age 〉 6 months, median 0. 66 （0. 12 - 3.52） mmol/mol creatinine. The concentration of urinary pipecolic acid was different between two groups of control （Z = -5. 464, P 〈 0. 01 ）. Twelve patients were all older than 6 months when they were tested, and the concentration was only mildly elevated in one patient, the range of 12 patients was 0. 14 - 4. 08 mmol/mol creatinine. The concentration was not significantly different between the control with age 〉 6 months and our PDE patients （Z = - 0. 655, P 〉 0. 05 ）. There were no significant correlations between the concentration of urinary pipecolic acid and the initial dose or maintenance dose at last follow-up of pyridoxine （ r = 0. 418 and 0. 166, P = 0.176 and 0. 607 ）. Conclusion Seizures start in early infancy in most PDE patients. The splicing mutation IVS11 ＋ 1G 〉 A is supposed to be a probable ＂hotspot＂ mutation with a high frequency in Chinese PDE patients. Most patients have different levels of psychomotor development delay after seizures are controlled, and the patients with birth abnormalities may have worse outcomes. No relationships between the diagnostic delay time and the development outcome are found. The concentration of urinary pipecolic acid can return to normal during treatment with pyridoxine.