Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing
Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing
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DOI：
10.1038/ejhg.2013.175
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发表时间：
2014-04-01
影响因子：
5.2
通讯作者：
Estivill, Xavier
中科院分区：
文献类型：
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作者：
Trujillano, Daniel;Perez, Belen;Estivill, Xavier
苯丙酮尿症（PKU）和四氢生物蝶呤（BH 4）缺陷型高苯丙氨酸血症（BH 4 DH）的遗传诊断依赖于扫描已知突变的方法或使用桑格测序的费力的分子工具。我们已经实施了一种新的和更有效的策略，基于高通量的多重靶向重测序的四个基因（PAH，GCH 1，PTS和QDPR），当受到功能丧失突变的影响时，导致PKU和BH 4DH。我们已经在95个具有先前已知的PAH、GCH 1、PTS和QDPR突变的样本和一个对照样本的队列中验证了这种方法。使用定制的NimbleGen SeqCap EZ Choice阵列富集合并的条形码化DNA文库，并使用HiSeq 2000测序仪测序。几种强大的生物信息学工具的组合使我们能够检测95个样本中所有已知的致病性突变（点突变，短插入/缺失和大基因组重排），而不会在对照样本中检测到这些基因中的虚假调用。然后，我们使用MiSeq测序仪在11名未表征的HPA患者的发现队列中使用相同的捕获测定。此外，我们报告了PAH中四个基因组重排的断点的精确表征，包括内含子3中899 bp的新缺失。我们的研究是一个原理证明，高通量靶向重测序准备取代经典的分子方法来进行高苯丙氨酸血症的差异遗传诊断，允许在出生后几天建立专门定制的治疗方法。
Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth.