Identification of Disease-Causing Mutations in Autosomal Dominant Retinitis Pigmentosa (adRP) Using Next-Generation DNA Sequencing
Identification of Disease-Causing Mutations in Autosomal Dominant Retinitis Pigmentosa (adRP) Using Next-Generation DNA Sequencing
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DOI：
10.1167/iovs.10-6180
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发表时间：
2011-01-01
影响因子：
4.4
通讯作者：
Daiger, Stephen P.
中科院分区：
文献类型：
--
作者：
Bowne, Sara J.;Sullivan, Lori S.;Daiger, Stephen P.
目的.确定大规模并行下一代DNA测序是否能快速有效地检测单基因遗传病患者的致病突变。视网膜色素变性（RP）是这项技术的一个具有挑战性的应用，因为它是一种单基因疾病的个人和家庭，但在患者群体中是高度异质性的。RP具有多种遗传模式，每种遗传模式在许多基因中具有突变，并且在每个位点具有许多不同的致病突变;此外，许多RP基因尚未被鉴定。下一代测序被用来确定突变对受影响的个人从21个家庭与常染色体显性遗传RP，从一个队列的家庭没有突变的“共同”RP基因。用454 GS FLX Titanium（Roche Diagnostics，Indianapolis，IN）和GAIIx（Illumina/Solexa，San Diego，CA）平台对靶向46个候选基因的249，267个独特碱基的1000个扩增子进行测序。454 GS FLX和GAIIx平台的平均序列深度分别为70 X和125 X。鉴定和分析了9000多个序列变异，以评估致病性的可能性。其中112人被选为可能的候选人，并与传统的双脱氧毛细管电泳测序的其他家庭成员和对照组的隔离测试。在21个家系中发现5个致病突变（24%）。该项目表明，下一代测序是检测引起异质性单基因疾病（如RP）的新型罕见突变的有效方法。随着这项技术的增加，现在可以在65%的常染色体显性遗传RP病例中鉴定出致病突变。（Invest Ophthalmol维斯科学。2011;52：494-503）DOI：10.1167/iovs.10-6180
PURPOSE. To determine whether massively parallel next-generation DNA sequencing offers rapid and efficient detection of disease-causing mutations in patients with monogenic inherited diseases. Retinitis pigmentosa (RP) is a challenging application for this technology because it is a monogenic disease in individuals and families but is highly heterogeneous in patient populations. RP has multiple patterns of inheritance, with mutations in many genes for each inheritance pattern and numerous, distinct, disease-causing mutations at each locus; further, many RP genes have not been identified yet.METHODS. Next-generation sequencing was used to identify mutations in pairs of affected individuals from 21 families with autosomal dominant RP, selected from a cohort of families without mutations in "common" RP genes. One thousand amplicons targeting 249,267 unique bases of 46 candidate genes were sequenced with the 454GS FLX Titanium (Roche Diagnostics, Indianapolis, IN) and GAIIx (Illumina/Solexa, San Diego, CA) platforms.RESULTS. An average sequence depth of 70X and 125X was obtained for the 454GS FLX and GAIIx platforms, respectively. More than 9000 sequence variants were identified and analyzed, to assess the likelihood of pathogenicity. One hundred twelve of these were selected as likely candidates and tested for segregation with traditional di-deoxy capillary electrophoresis sequencing of additional family members and control subjects. Five disease-causing mutations (24%) were identified in the 21 families.CONCLUSION. This project demonstrates that next-generation sequencing is an effective approach for detecting novel, rare mutations causing heterogeneous monogenic disorders such as RP. With the addition of this technology, disease-causing mutations can now be identified in 65% of autosomal dominant RP cases. (Invest Ophthalmol Vis Sci. 2011;52:494-503) DOI:10.1167/iovs.10-6180