摘要目的 研究1例临床疑诊尼曼-匹克病C型(Niemann-Pick disease type C,NPC)先证者的基因诊断,以及其家系的产前基因诊断.方法 采用DNA抽提试剂盒提取先证者(NPC晚发婴儿型)和其父母外周血白细胞基因组DNA,采用聚合酶链反应-直接测序法进行NPC1基因突变检测.测序结果与GenBank人类NPC1基因(NM 000271)进行比较,突变外显子片段均行DNA双向测序证实.同时选取50例正常人作为对照,通过直接测序法以排除新错义突变为基因多态性.采用ClustalX 1.81分析软件对人类和其他种属的NPC1氨基酸序列进行同源性比较.在先证者母亲再次妊娠18周时抽取羊水细胞,对培养后羊水细胞进行相应的基因突变检测,用于产前基因诊断.抽取新生儿外周血白细胞基因组DNA进行NPC1基因分析.结果 NPC1基因测序发现先证者携带2个新杂合突变c.2284-2287 delCTCT和p.V959G,分别来源于父亲和母亲,50例正常人中未发现这2种突变.通过ClustalX 1.81软件比对人、小鼠、大鼠、兔、猫及猪的NPC1蛋白的氨基酸发现,p.V959为中度保守氨基酸区域,此位点氨基酸错义突变p.V959G可能导致NPC蛋白功能异常.胎儿羊水直接抽提基因组DNA及培养后羊水细胞DNA检测均未发现这2种突变,为正常胎儿.新生儿外周血白细胞的NPC1基因分析未发现携带c.2284-2287 delCTCT和p.V959G突变,与产前基因诊断结果相符.结论 聚合酶链反应-直接测序法可为NPC先证者进行基因诊断,也可为其家系进行产前基因诊断,p.V959突变可能与晚期婴儿型临床表型相关.

abstractsObjective To analyze gene mutations of a Niemann-Pick disease type C (NPC) proband,and carry out prenatal diagnosis for the family.Methods The coding regions of NPC1 gene in the proband (late-infantile form) and white blood cell (WBC) in peripheral blood of its parents were amplified by polymerase chain reaction and direct DNA sequencing in both directions was performed.The sequencing results were compared with human NPC1 gene sequence (NM_000271) in GenBank,and sequences of mutated exons were determined.Direct sequencing was used on 50 normal Chinese individuals' DNA samples (control) to exclude mutation's single nucleotide polymorphism (SNP).An inter-species alignment of homologous NPC1 proteins was performed using ClustalX 1.81 software.During the second pregnancy of the proband's mother,the amniotic fluid was obtained at 18 weeks of gestation and the amniocytes were cultured for gene mutation analysis.Neonate's DNA of WBC in peripheral blood was also extracted for NPC1 gene analysis.Results Mutation analysis of NPC1 gene revealed two novel heterozygous mutations (c.2284-2287 delCTCT and p.V959G) in the proband,which originated from her father and mother,respectively.These two mutations were absent in the control,suggesting that these mutations were not SNP.While comparing with the amino acid in NPC1 protein of human,mouse,rat,rabbit,cat and pig,it revealed that p.V959 belonged to a conservative amino acid region and the missense mutation of p.V959G may perturb the function of NPC protein.Neither mutation was found in DNA from amniotic fluid or from the cultivated amniocytes in the second pregnancy,suggesting a normal fetus.c.2284-2287 delCTCT and p.V959G mutation were not found in NPC1 gene analysis of WBC in peripheral blood of the neonate,which was consistent with the prenatal diagnosis.Conclusions PCR-direct sequencing could be used as genetic diagnosis for NPC proband and prenatal diagnosis for its family.The mutation p.V959G may be correlated to late infantile form of NPC.