CAJ | 학술논문

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.
목적 연구1례림상의진니만-필극병C형(Niemann-Pick disease type C,NPC)선증자적기인진단,이급기가계적산전기인진단.방법 채용DNA추제시제합제취선증자(NPC만발영인형)화기부모외주혈백세포기인조DNA,채용취합매련반응-직접측서법진행NPC1기인돌변검측.측서결과여GenBank인류NPC1기인(NM 000271)진행비교,돌변외현자편단균행DNA쌍향측서증실.동시선취50례정상인작위대조,통과직접측서법이배제신착의돌변위기인다태성.채용ClustalX 1.81분석연건대인류화기타충속적NPC1안기산서렬진행동원성비교.재선증자모친재차임신18주시추취양수세포,대배양후양수세포진행상응적기인돌변검측,용우산전기인진단.추취신생인외주혈백세포기인조DNA진행NPC1기인분석.결과 NPC1기인측서발현선증자휴대2개신잡합돌변c.2284-2287 delCTCT화p.V959G,분별래원우부친화모친,50례정상인중미발현저2충돌변.통과ClustalX 1.81연건비대인、소서、대서、토、묘급저적NPC1단백적안기산발현,p.V959위중도보수안기산구역,차위점안기산착의돌변p.V959G가능도치NPC단백공능이상.태인양수직접추제기인조DNA급배양후양수세포DNA검측균미발현저2충돌변,위정상태인.신생인외주혈백세포적NPC1기인분석미발현휴대c.2284-2287 delCTCT화p.V959G돌변,여산전기인진단결과상부.결론 취합매련반응-직접측서법가위NPC선증자진행기인진단,야가위기가계진행산전기인진단,p.V959돌변가능여만기영인형림상표형상관.
Objective 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.