Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA
Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA
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DOI：
10.1111/jth.12339
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发表时间：
2013-09-01
影响因子：
10.4
通讯作者：
El-Maarri, O.
中科院分区：
文献类型：
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作者：
Pezeshkpoor, B.;Zimmer, N.;El-Maarri, O.
SummaryBackgroundIn一小群典型的血友病A（HA）患者没有突变的F8编码序列（cDNA）could be found. In目前的研究中，我们进行了系统的筛选与减少FVIII：C水平的遗传和非遗传参数在一组大多是轻度HA（只有一个中度）患者没有检测到突变的F8 cDNA.MethodsWe确定FVIII和VWF活性和抗原水平，并进行VWF-FVIII结合（VWF：FVIIIB）和VWF-胶原结合试验（VWF：CB）以及VWF多聚体分析。对VWF进行完全测序以排除突变。使用重叠长距离PCR（LR-PCR）结合下一代测序（NGS）方法对F8基因座（包括内含子）进行测序。此外，F8 mRNA定量和定性实时PCR（qRT）和重叠反转录（RT）PCRs.ResultsAll VWF测试是正常的。LR-PCR证明了F8基因座的完整性。在患者中发现了八种独特的多态性，其中两种是复发性的。此外，RT-PCR分析证实，两个独特的变体创建可检测到的新的隐蔽剪接位点的患者，导致在引入的内含子DNA序列到mRNA和创建过早的终止codons.ConclusionBy系统地排除所有可能的原因HA，我们可以非常肯定地得出结论，深内含子突变F8，虽然罕见，导致异常的mRNA剪接，导致轻度HA。
SummaryBackgroundIn a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non-genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA.MethodsWe determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LR-PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively.ResultsAll VWF tests were normal. The LR-PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons.ConclusionBy systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.