Allelic copy number variation in FSCN2 detected using allele-specific genotyping and multiplex real-time PCRs
Allelic copy number variation in FSCN2 detected using allele-specific genotyping and multiplex real-time PCRs
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DOI：
10.1167/iovs.07-1656
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发表时间：
2008-09-01
影响因子：
4.4
通讯作者：
Takahashi, Masayo
中科院分区：
文献类型：
--
作者：
Jin, Zi-Bing;Mandai, Michiko;Takahashi, Masayo
目的。等位基因拷贝数变异（CNV）可能会改变杂合突变的功能效应。然而，其在遗传性疾病中的潜在机制及其作用在很大程度上尚不清楚。在本研究中，对已报道的 FSCN2 突变进行了检查，该突变不仅存在于色素性视网膜炎 (RP) 患者中，而且存在于正常人群中。方法。进行实验以调查具有 c 的 RP 患者 FSCN2 的基因和等位基因拷贝数。 72delG 突变以及有或没有该突变的健康受试者。建立了基于实时 PCR 的基因分型方法，该方法使用实时 PCR 测定来鉴定 FSCN2 基因野生型和突变等位基因的拷贝数。 结果。三名 RP 患者和三名正常受试者的等位基因比例相等。有趣的是，另一名患者的野生型等位基因与突变型等位基因的拷贝数比例不对称（4:1）。使用定量测定进一步验证了这些发现。等位基因特异性甲基化测定表明 FSCN2 基因中存在随机甲基化模式。结论。对突变样品中 FSNC2 基因和每个等位基因的拷贝数进行了定量。研究结果排除了等位基因 CNV 与 RP 相关的可能性，表明 c。 72delG 变异不是 RP 的主要原因。 FSCN2 基因不太可能有差异印记。本研究开发的基于实时 PCR 的基因分型方法对于研究 CNV 基因组区域内的等位基因不对称性非常有用。
PURPOSE. Allelic copy number variation (CNV) may alter the functional effects of a heterozygous mutation. The underlying mechanisms and their roles in hereditary diseases, however, are largely unknown. In the present study an FSCN2 mutation was examined that has been reported, not only in patients with retinitis pigmentosa (RP), but also in the normal population.METHODS. Experiments were performed to investigate the gene and allele copy numbers of FSCN2 in patients with RP who have the c. 72delG mutation as well as healthy subjects with or without the mutation. A real-time PCR-based genotyping approach was established that used a real-time PCR assay to qualify the copy numbers of both the wild-type and mutant alleles of the FSCN2 gene.RESULTS. Three patients with RP and three normal subjects had an equal ratio of the alleles. Of interest, another patient had an asymmetric allele ratio (4: 1) of the copy number of the wildtype allele, compared with that of the mutant allele. These findings were further verified using quantitative assays. An allele-specific methylation assay demonstrated a random methylation pattern in the FSCN2 gene.CONCLUSIONS. The copy numbers of the FSNC2 gene and of each allele in the mutant samples were quantified. The findings excluded the possibility that allelic CNV was associated with RP, suggesting that the c. 72delG variant is not the primary cause of RP. It is not likely that the FSCN2 gene is imprinted differentially. The real-time PCR-based genotyping method developed in this study is useful for investigations of allelic asymmetries within genomic regions with CNVs.