School of Medicine, Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan.
Division of Pediatric Hematology/Oncology, Hemophilia Center, Taipei Medical University Hospital, Taipei, Taiwan.
Eur J Haematol. 2019 Jul;103(1):47-55. doi: 10.1111/ejh.13242. Epub 2019 May 21.
In 10%-18% of mild-type hemophilia A (HA) patients, mutations cannot be found by routine DNA analysis.
We aimed to identify the genetic defects by mRNA analysis of F8 gene in mild HA patients without mutation in exonic DNA.
From 2006 to 2016, we identified F8 exon mutations in 39 of 49 mild HA patients using routine genetic testing. We then evaluated the 10 remaining patients from six unrelated families without exonic DNA mutation by performing cDNA sequence analysis.
Nine of the 10 (90%) patients were confirmed to have F8 gene mutation. Eight patients from four unrelated families were notably found to have presence of an aberrant 675-bp fragment. Sequencing of this fragment showed that there were two separate new alternative splicing exons of 35 bp and 55 bp within intron 18, which formed a 90-bp insertion between exon 18 and exon 19 (E18ins90bpE19) in the mRNA. Based on direct sequencing, this alternative splicing transcript appears to have resulted from deep intronic variant c.5999-277G>A of intron 18.
Our study suggests that deep intronic variant of c.5999-277G>A may be a hot spot mutation for mild hemophilia patients without mutation in exonic DNA.
在 10%-18%的轻度 A 型血友病（HA）患者中，常规 DNA 分析无法发现突变。
我们旨在通过对无外显子 DNA 突变的轻度 HA 患者的 F8 基因 mRNA 分析来确定遗传缺陷。
2006 年至 2016 年，我们通过常规基因检测在 49 名轻度 HA 患者中鉴定了 39 名患者的 F8 外显子突变。然后，我们通过 cDNA 序列分析评估了来自六个无关家庭的另外 10 名无外显子 DNA 突变的患者。
10 名患者中的 9 名（90%）被证实存在 F8 基因突变。来自四个无关家庭的 8 名患者明显存在异常的 675-bp 片段。对该片段进行测序表明，在 18 号内含子内存在两个单独的新的 35-bp 和 55-bp 外显子替代剪接，在 18 号外显子和 19 号外显子之间形成了一个 90-bp 的插入（E18ins90bpE19）。基于直接测序，这种替代剪接转录物似乎是由 18 号内含子的 c.5999-277G>A 深内含子变异引起的。
我们的研究表明，c.5999-277G>A 的深内含子变异可能是无外显子 DNA 突变的轻度血友病患者的热点突变。
