Characterization of two large duplications of F9 associated with mild and severe haemophilia B, respectively
Characterization of two large duplications of F9 associated with mild and severe haemophilia B, respectively
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DOI：
10.1111/hae.13704
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发表时间：
2019-05-01
期刊：
影响因子：
3.9
通讯作者：
Ding, Qiulan
中科院分区：
文献类型：
--
作者：
Xie, Xiaoling;Chen, Changming;Ding, Qiulan
只有两个大的F9重复引起血友病B（HB）的报道。目的分析F9基因大重复突变的致病机制。方法我们分别在先证者A和B中发现了与轻度和重度HB相关的两个F9大重复（dupex 1-6和dupex 4-6）。在这里，我们使用长距离PCR和基因组步移结合定量引物步移策略定位两个重复的断点。通过单倍型分析追溯了dup ex 4-6的来源，并对散发家系B进行了体细胞嵌合体检测，通过微基因分析检测了重复产生的嵌合内含子对转录的影响。结果分叉停滞、模板转换和/或微同源介导的断裂诱导复制（FoSTeS/MMBIR）机制可能是形成两个串联直接复制的原因。先证者B为体细胞嵌合型和生殖系嵌合型，重复基因4-6可追溯至其外祖母，可能是在其早期胚胎细胞有丝分裂过程中形成的。Minigene分析表明嵌合内含子产生三个转录本，一个次要转录本产生在正常FIX中添加重复的143个氨基酸的框内蛋白，解释了Western blot中显示的少量较大FIX。与原始F9拷贝相邻的inter-F9 dupex 1-6产生了两个相同的启动子，启动子竞争可能是导致轻度HB的复制的致病机制。结论重复基因可能通过复杂的致病机制与疾病相关。
Introduction Only two large duplications of F9 causing haemophilia B (HB) have been reported. Aim To analyse the pathogenic mechanisms of large F9 duplications. Methods We have identified two large duplications of F9 (dup ex 1-6 and dup ex 4-6) associated with mild and severe HB in probands A and B, respectively. Here, we localized the breakpoints of the two duplications using long-range PCR and genome walking combined with quantitative primer walking strategies. We traced the origin of dup ex 4-6 by haplotype analysis then performed somatic mosaicism detection in sporadic pedigree B and detected the effect of chimeric intron derived from the duplication on transcription by minigene assay. Results Mechanisms of fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) might be responsible for the formation of two tandem direct duplications. The dup ex 4-6 was traced to maternal grandmother of proband B, who was both somatic mosaicism and germline mosaic and the duplication might be formed during mitosis of her early embryonic cells. Minigene assay demonstrated that chimeric intron generated three transcripts, one minor transcript produced an in-frame protein adding duplicated 143 amino acids into the normal FIX, explaining the small amount of larger FIX shown in Western blot. The inter-F9 dup ex 1-6 adjacent to the original F9 copy created two identical promoters, and promoter competition might be the pathogenic mechanism of the duplication causing mild HB. Conclusions This study highlights that duplications can be associated with diseases by complicated pathogenic mechanisms.