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目的研究2个无关的A型尼曼-匹克病家系酸性神经鞘磷脂酶(ASM)基因SMPD1突变及遗传特征,分析基因型与表型关系。方法收集2位先证者及其家庭成员的临床资料,采用基因组小剂量抽提试剂盒从2个家系共6名成员及100例健康对照者外周血中提取基因组DNA,根据人类基因组数据库中获得的基因序列(NM000543)设计4对引物,采用聚合酶链反应(PCR)进行扩增,并应用PCR产物回收试剂盒进行产物回收,采用DNA直接测序法进行基因突变检测,测序结果应用DNAman软件进行序列对比分析,确定基因突变位点,对测序异常的片段重新进行PCR扩增与测序,以验证结果的可靠性。结果 PCR扩增所得片段与预计扩增片段大小一致,无非特异扩增带,可以进行纯化与测序。将测序结果与正常序列进行对比分析后,在先证者1和2的SMPD1基因的第1号外显子均发现一个纯合突变T107C,遗传密码子由GTG变为GCG,从而导致36位缬氨酸被丙氨酸替代(p.V36A),最终致使ASM(E.C.3.1.4.12)活性缺陷,产生临床症状。先证者之父母为携带者,100例健康对照中未发现上述突变。结论证实SMPD1基因是本研究中2个无关的A型尼曼-匹克病家系的致病基因。2个家系患儿均为SMPD1基因纯合突变致病,其父母为表型正常的基因突变携带者。
Objective To investigate the mutation and genetic characteristics of SMPD1 gene in two unrelated serotypes of sphingomyelinase (ASM) in type A Nehre-Pick family with type A and analyze the relationship between genotype and phenotype. Methods The clinical data of two probands and their family members were collected. Genomic DNA was extracted from the peripheral blood of 6 members of 2 pedigrees and 100 healthy controls using genome-wide low-dose extraction kit. According to the results obtained from the human genome database (NM000543). Four pairs of primers were designed and amplified by polymerase chain reaction (PCR). The products were recovered by PCR product recovery kit. DNA sequencing was used to detect the gene mutation. The DNA sequencing results were obtained by DNAman software Sequence comparative analysis to determine the site of gene mutation, sequencing of abnormal fragments were re-PCR amplification and sequencing to verify the reliability of the results. Results The PCR amplified fragment was consistent with the size of the expected amplified fragment, non-specific amplification band, can be purified and sequenced. After comparing the sequencing results with normal sequences, a homozygous mutant T107C was found in exon 1 of SMPD1 gene of probands 1 and 2, and the genetic code was changed from GTG to GCG, resulting in the formation of 36-val The acid is replaced by alanine (p.V36A), eventually resulting in ASM (EC 3.1.4.12) activity deficiencies, resulting in clinical symptoms. The parents of probands were carriers, and no mutation was found in 100 healthy controls. Conclusion The SMPD1 gene was confirmed to be the causative gene of two unrelated type A Niemann-Pick families in this study. Two families of children with SMPD1 gene homozygous mutation pathogenic, the parents of phenotypic normal gene mutation carriers.