Deep intronic variant c.5999‐277G>A of F8 gene may be a hot spot mutation for mild hemophilia A patients without mutation in exonic DNA

BACKGROUND: In 10%-18% of mild-type hemophilia A (HA) patients, mutations cannot be found by routine DNA analysis. OBJECTIVE: We aimed to identify the genetic defects by mRNA analysis of F8 gene in mild HA patients without mutation in exonic DNA. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.