Leber congenital amaurosis - A model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson memorial lecture
Leber congenital amaurosis - A model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson memorial lecture
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DOI：
10.1016/j.ajo.2007.08.022
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发表时间：
2007-12-01
影响因子：
4.2
通讯作者：
Stone, Edwin M.
中科院分区：
文献类型：
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作者：
Stone, Edwin M.
目得：批判性地评价我们在莱伯先天性黑蒙（LCA）分子检测方面的经验，并利用这些信息设计一种通用的方法来治疗异质性隐性疾病。仔细的临床和分子特征的大队列患者的影响与遗传性眼病将是一个重要的一步，在发展有效的治疗这些疾病，特别是当治疗涉及基因replacement.Design：一个分子遗传学的情况下，对照研究。六百四十，对两名临床诊断为LCA的无关个体和200名无关对照个体进行疾病筛查，使用单链构象多态性分析（SSCP）、自动DNA测序、多重等位基因特异性连接分析（SNPs）和高密度固相单核苷酸多态性基因分型的各种组合在8个基因中引起序列变异。在这项研究中观察到189种不同的致病序列变异的440例，其中98例以前没有报道过。189个变异中有146个（77%）仅在单个个体中观察到。观察到的变异在LCA患者或八个基因中分布不均匀。对这种不均匀分布的经验分析用于设计多平台突变检测策略，其比对所有受试者中所有LCA基因的所有编码区进行完全测序的更常规策略效率高四倍。Hardy-Weinberg对观察到的突变的分析表明，这八个基因共同负责北美约70%的LCA病例。发现最常见的LCA等位基因（CEP 290中的内含子26变异）的携带者频率为2/3，248，这表明该人群中LCA的总体患病率约为1/81，000。设计了等位基因特异性连接试验（SNALLY）来检测68个最常见的导致LCA的等位基因，并且对来自该试验的数据的半定量分析还揭示了队列中基因缺失和同二体性的实例。数据表明，分层筛选策略结合等位基因-与单独DNA测序相比，自动DNA测序的特异性检测可以将常染色体隐性突变检测的效率提高四倍。然而，本研究中观察到的非常高的独特突变率（77%）表明，如果要实现高灵敏度，DNA测序仍将是整体策略的重要组成部分。
PURPOSE: To critically evaluate our experience in molecular testing of Leber congenital amaurosis (LCA) and to use this information to devise a general approach to heterogeneous recessive disorders. Careful clinical and molecular characterization of large cohorts of patients affected with inherited eye diseases will be an essential step in the development of effective therapy for these diseases, especially when the therapy involves gene replacement.DESIGN: A molecular genetic case,control study.METHODS: Six hundred forty,two unrelated individuals with the clinical diagnosis of LCA and 200 unrelated control individuals were screened for disease-causing sequence variations in eight genes using various combinations of single,strand conformational polymorphism analysis (SSCP), automated DNA sequencing, multiplex allele specific ligation analysis (SNPlex), and high,density solid-phase single nucleotide polymorphism genotyping.RESULTS: Four hundred forty instances of 189 different disease-causing sequence variations were observed in this study, 98 of which have not been previously reported. One hundred forty,six of the 189 variations (77%) were observed in only a single individual. The observed variations were not evenly distributed among the LCA patients or among the eight genes. Empirical analysis of this uneven distribution was used to devise a multi,platform mutation detection strategy that is four times more efficient than a more conventional strategy of completely sequencing all of the coding regions of all LCA genes in all subjects. Hardy-Weinberg analysis of the observed mutations suggests that these eight genes are collectively responsible for about 70% of the cases of LCA in North America. The carrier frequency of the most common LCA allele (an intron 26 variation in CEP290) was found to be 2/3,248, which suggests that the overall prevalence of LCA in this population is about 1/81,000. An allele-specific ligation assay (SNPlex) was designed to detect 68 of the most common LCA-causing alleles, and semi,quantitative analysis of the data from this assay also revealed examples of gene deletion and isodisomy in the cohort.CONCLUSIONS: The data demonstrates that a tiered screening strategy combining allele-specific detection with automated DNA sequencing can increase the efficiency of autosomal recessive mutation detection fourfold when compared with DNA sequencing alone. However, the very high rate of unique mutations ob, served in this study (77%) suggests that DNA sequencing will remain an important part of the overall strategy if high sensitivity is to be achieved.