探讨基因分析联合蛋白质印迹(Western blotting)法在极长链酰基辅酶A脱氢酶缺乏症(VLCADD)ACADVL基因新发突变位点致病性鉴定中的临床应用。

收集2017年6月1日至2020年10月31日南京市新生儿疾病筛查中心筛查的290 738例新生儿遗传代谢病筛查样本中，通过串联质谱法检测疑似VLCADD的4例患儿及父母，采集外周血进行基因分析，并用Western blotting检测外周血中极长链酰基辅酶A脱氢酶(VLCAD，也称为ACADVL)蛋白表达量变化。

4例患儿通过基因分析被诊断为VLCADD，共检测到8个突变位点[2个疑似致病变异(c.664 G>C、c.342+1 G>A)，5个临床意义不明(c.694 G>A、c.1699 C>T、c.1030 A>G、c.1247 C>T、c.833_835 del)，1个位点为首次报道(c.1077+6 T>A)]。4个家系共计12个外周血样本通过Western blotting均能检测到ACADVL蛋白的表达，但患儿的目标蛋白表达量均明显低于其父母。家系1的外周血样本保存时间较另外3个家系长，ACADVL蛋白在家系1的父母检测中明显低于另外3个家系；且与外周血保存时间基本一致的非VLCADD家系5比较，家系5内参ACTIN蛋白表达量下降更为明显。

疑似VLCADD患儿的ACADVL基因检测的位点为临床意义不明时，建议使用新鲜的外周血样本，通过Western blotting完善ACADVL蛋白检测，该结果与基因检测结果可互为补充。

To explore the clinical application of genetic analysis combined with Western blotting in the identification of the pathogenicity of novel mutations in ACADVL gene of very long chain acyl-CoA dehydrogenase deficiency (VLCADD).

From June 1, 2017 to October 31, 2020, 290 738 blood samples from newborns screened in Nanjing Neonatal Disease Screening Center were analysed. Among them, 4 newborns were suspected of VLCADD by tandem mass spectrometry. Peripheral blood was collected from the 4 newborns and their families for gene analysis, and the expression of very long chain acyl-CoA dehydrogenase (VLCAD, ACADVL) in peripheral blood was detected by Western blotting.

Four children were diagnosed as VLCADD through gene analysis, and 8 mutation sites were detected: 2 likely pathogenic variants (c.664 G>C、c.342+ 1 G>A), 5 novel variants (c.694 G>A、c.1699 C>T、c.1030 A>G、c.1247 C>T、c.833_835 del), and 1 site was the first report (c.1077+ 6 T>A). A total of 12 peripheral blood samples from the 4 families were collected to detect the expression of ACADVL protein by Western blotting. The results confirmed that the target protein expressions of the 4 patients were significantly lower than those of their parents. The storage time of the peripheral blood samples in family 1 was longer than that of other three families, accordingly the ACADVL protein level in family 1 were significantly lower than that of other three families. Compared with family 1, non-VLCADD family 5 with the same storage time of peripheral blood, the decrease of internal reference ACTIN protein was more obvious.

When the detection site of ACADVL gene in children with suspected VLCADD is of unclear clinical significance, it is recommended to use fresh peripheral blood samples to detect the ACADVL protein by Western blotting, the results of which can be complementary with the genetic results.