背景:尼曼-匹克病C型(NPC)是一种罕见的溶酶体神经内脏贮积病,由NPC1(95%)或NPC2(5%)基因突变引起。NPC1和NPC2基因的产物在糖脂和胆固醇运输中起重要作用,因此,这可能导致具有各种表型的NPC疾病,这些表型表现出广泛的症状。
方法:在本研究中,纳入了35名伊朗NPC无关患者。这些患者首先通过用于NPC诊断的细胞中胆固醇沉积物的Filipin染色测试进行分析。按照制造商的方案从EDTA中的外周血白细胞样品中提取基因组DNA。使用适当的引物组通过聚合酶链反应(PCR)扩增NPC1基因的所有外显子-内含子边界和编码外显子。此后,使用NCBI数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)。通过包括人类基因突变数据库(HGMD)(http://www.hgmd.cf.AC.英国/交流/指数。php)和ClinVar(https://www.ncbi.nlm.nih.gov/clinvar(.此外,根据美国医学遗传学和基因组学学院(ACMG)指南对所有变异体进行人工分类.
结果:序列分析揭示了20种不同的变异,其中10个是新的,包括一个无义突变(c.406.C>T);三个小缺失,(c.3126delC,c.2920_2923delCCTG,和c.2037delG);和六个可能的致病性错义突变,(c.542C>A,c.1970G>A,c.1993C>G,c.2821T>C,c.2872C>G,和c.3632T>A)。最后,使用ACMG指南确定这些新变异体的致病性.
结论:本研究旨在促进未来NPC患者的产前诊断。在这方面,我们发现了10个新的突变,并验证了它们中的大多数发生在六个NPC1外显子(5、8、9、13、19和21)中,这应该被视为伊朗患者的高度优先考虑的成本效益评估。
BACKGROUND: Niemann-Pick disease type C (NPC) is a rare lysosomal neurovisceral storage disease caused by mutations in the NPC 1 (95%) or NPC2 (5%) genes. The products of NPC1 and NPC2 genes play considerable roles in glycolipid and cholesterol trafficking, which could consequently lead to NPC disease with variable phenotypes displaying a broad spectrum of symptoms.
METHODS: In the present study 35 Iranian NPC unrelated patients were enrolled. These patients were first analysed by the Filipin Staining test of cholesterol deposits in cells for NPC diagnostics. Genomic DNA was extracted from the samples of peripheral blood leukocytes in EDTA following the manufacturer\'s protocol. All exon-intron boundaries and coding exons of the NPC1gene were amplified by polymerase chain reaction (PCR) using appropriate sets of primers. Thereafter, the products of PCR were sequenced and analysed using the NCBI database ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The variants were reviewed by some databases including the Human Gene Mutation Database (HGMD) ( http://www.hgmd.cf.ac.uk/ac/index.php ) and ClinVar ( https://www.ncbi.nlm.nih.gov/clinvar (. Moreover, all the variants were manually classified in terms of the American College of Medical Genetics and Genomics (ACMG) guideline.
RESULTS: The sequence analysis revealed 20 different variations, 10 of which are new, including one nonsense mutation (c.406C > T); three small deletions, (c.3126delC, c.2920_2923delCCTG, and c.2037delG); and six likely pathogenic missense mutations, (c.542C > A, c.1970G > A, c.1993C > G, c.2821 T > C, c.2872C > G, and c.3632 T > A). Finally, the pathogenicity of these new variants was determined using the ACMG guidelines.
CONCLUSIONS: The present study aimed to facilitate the prenatal diagnosis of NPC patients in the future. In this regard, we identified 10 novel mutations, and verified that the majority of them occurred in six NPC1 exons (5, 8, 9, 13, 19, and 21), that should be considered with a high priority for Iranian patients\' cost-effective evaluation.