The utility of massively parallel sequencing for posterior polymorphous corneal dystrophy type 3 molecular diagnosis
The utility of massively parallel sequencing for posterior polymorphous corneal dystrophy type 3 molecular diagnosis
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DOI：
10.1016/j.exer.2019.03.002
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发表时间：
2019-05-01
影响因子：
3.4
通讯作者：
Liskova, Petra
中科院分区：
文献类型：
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作者：
Dudakova, Lubica;Evans, Cerys J.;Liskova, Petra
本研究的目的是确定不同来源的后多形性角膜营养不良（PPCD）先证的分子遗传原因，并评估大规模平行测序在检测ZEB1突变中的效用。我们总共调查了12个家庭（5个英国家庭、4个捷克家庭、1个斯洛伐克家庭和2个瑞士家庭）。通过Sanger （n = 5）、外显子组（n = 4）和基因组（n = 3）测序，在先显子中鉴定出10个新的ZEB1致病突变和2个复发性突变。Sanger测序用于确认大规模平行测序检测到的突变，并进行分离分析。基因组测序显示，一个先证者在1-7外显子和部分8外显子上发现了一个新的类似于0.34 Mb的杂合从头缺失。转录本分析证实，ZEB1转录本在血液来源的RNA样本中可检测到，并且疾病相关变异c.482-2A > G导致异常的pre-mRNA剪接。新发突变是PPCD3的一个特征，在目前的研究中发现，其发生率至少为16.6%。一般来说，大规模平行测序是检测ppcd3相关突变的一种省时的方法，重要的是，基因组测序能够识别完全或部分杂合的ZEB1缺失，这些缺失可以逃避Sanger和外显子组测序的检测。这些发现有助于我们对PPCD3的理解，目前已经鉴定出49种致病变异，所有这些变异都被预测为无效等位基因。
The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel similar to 0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A > G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles.