肉碱缺乏症性心肌病患儿血浆循环游离线粒体DNA定量分析及其临床意义

Quantitative analysis of circulating cell-free mitochondrial DNA in plasma of children with primary carnitine deficiency-associated cardiomyopathy and its clinical significance

目的检测肉碱缺乏症性心肌病患儿血浆循环游离线粒体DNA(ccf-mtDNA)水平并探讨其临床意义。方法前瞻性病例对照研究。收集2017年7月至2022年6月吉林大学第一医院小儿心血管科病房收治的7例以心肌病为主要临床表现的原发性肉碱缺乏症(PCD)患儿(PCD组)、同期就诊的16例扩张型心肌病(DCM)患儿(DCM组)及50例健康体检儿童(健康对照组)血液样本,采用实时荧光定量PCR方法检测各组血浆ccf-mtDNA水平并比较其差异,分析PCD组中血浆ccf-mtDNA水平与血游离肉碱水平、心功能的相关性,并监测PCD组应用左旋肉碱治疗后ccf-mtDNA的水平变化。3组间比较采用Kruskal-Wallis检验,PCD组与健康对照组比较采用Mann-Whitney检验,PCD组内治疗前后比较采用配对Wilcoxon秩和检验,变量间相关分析采用Logistic回归分析。结果PCD组、DCM组患儿血浆ccf-mtDNA水平分别为3.69×10^(6)(1.09×10^(6)~7.26×10^(6))拷贝/L、0.99×10^(6)(0.25×10^(6)~4.10×10^(6))拷贝/L,明显高于健康对照组[0.09×10^(6)(0.01×10^(6)~0.35×10^(6))拷贝/L](H=33.34、24.69,均P<0.001),且PCD组较DCM组更高(H=6.31,P<0.05)。PCD组应用左旋肉碱治疗前:血浆ccf-mtDNA水平与血游离肉碱水平、左心室射血分数呈负相关(r=-0.85、-0.82,均P<0.05),与改良Ross评分、N-末端B型利钠肽原水平呈正相关(r=0.81、0.83,均P<0.05)。PCD组应用左旋肉碱治疗后:血浆ccf-mtDNA水平逐渐下降,血游离肉碱水平、心脏功能逐渐恢复。血浆ccf-mtDNA水平自治疗第3个月[0.96×10^(6)(0.50×10^(6)~2.27×10^(6))拷贝/L]开始,较治疗前明显下降(Z=2.24,P<0.05),在第6、9及12个月分别为0.27×10^(6)(0.18×10^(6)~0.76×10^(6))拷贝/L、0.29×10^(6)(0.19×10^(6)~0.78×10^(6))拷贝/L及0.16×10^(6)(0.10×10^(6)~1.06×10^(6))拷贝/L,与健康对照组[0.09×10^(6)(0.01×10^(6)~0.35×10^(6))拷贝/L]比较差异均无统计学意义(Z=1.23、1.09、2.12,均P>0.05)。结论血浆ccf-mtDNA可能是肉碱缺乏症性心肌病的致病因素之一,其水平对此疾病的心脏病情评估具有一定的临床价值。

Objective To detect the level of the circulating cell-free mitochondrial DNA(ccf-mtDNA)in plasma of children with primary carnitine deficiency(PCD)-associated cardiomyopathy and evaluate its clinical significance.Methods In this prospective case-control study,peripheral blood samples were collected from 7 PCD patients with cardiomyopathy(PCD group),16 dilated cardiomyopathy(DCM)patients(DCM group),and 50 healthy children(healthy control group)in the Pediatric Cardiovascular Department Ward of First Hospital of Jilin University from July 2017 to June 2022.The ccf-mtDNA levels were measured and compared between groups by the real-time fluorescence quantitative polymerase chain reaction.The correlations between plasma ccf-mtDNA level and blood free carnitine level and cardiac function in the PCD group were analyzed.The changes in the ccf-mtDNA level were monitored after L-carnitine treatment in the PCD group.The Kruskal-Wallis test was used for comparison among the three groups.The Mann-Whitney test was used for comparison between the PCD group and the control group.Changes before and after treatment in the PCD group were analyzed using the paired Wilcoxon rank sum test.The correlation between variables was evaluated by Logistic regression.Results The plasma ccf-mtDNA levels in the PCD and DCM groups were 3.69×10^(6)(1.09×10^(6)-7.26×10^(6))copies/L and 0.99×10^(6)(0.25×10^(6)-4.10×10^(6))copies/L,respectively,which were significantly higher than that in the healthy control group[0.09×10^(6)(0.01×10^(6)-0.35×10^(6))copies/L](H=33.34,24.69;all P<0.01).Besides,the plasma ccf-mtDNA level in the PCD group was higher than that in the DCM group(H=6.31,P<0.05).In the PCD group,the plasma ccf-mtDNA level was negatively correlated with the blood free carnitine level and left ventricular ejection fraction(r=-0.85,-0.82,all P<0.05)and positively correlated with the modified Ross score and the N-terminal pro B type natriuretic peptide level(r=0.81,0.83,all P<0.05)before L-carnitine treatment.After treatment,the plasma ccf-mtDNA level decreased,and the blood free carnitine level and cardiac function recovered in the PCD group.The plasma ccf-mtDNA level declined sharply from the 3rd month[0.96×10^(6)(0.50×10^(6)-2.27×10^(6))copies/L]after treatment(Z=2.24,P<0.05)and got to 0.27×10^(6)(0.18×10^(6)-0.76×10^(6))copies/L,0.29×10^(6)(0.19×10^(6)-0.78×10^(6))copies/L,and 0.16×10^(6)(0.10×10^(6)-1.06×10^(6))copies/L at the 6th,9th,and 12th months after treatment,respectively,with no statistically significant difference compared to the healthy control group[0.09×10^(6)(0.01×10^(6)-0.35×10^(6))copies/L](Z=1.23,1.09,2.12;all P>0.05).Conclusions Plasma ccf-mtDNA may act as one pathogenic factor of cardiomyopathy in PCD,and monitoring its level is clinically important for heart condition assessment in PCD.