90-bp Insertion of Deep Intron between E18 and E19 (E18ins90bpE19) May Be a Hot-spot Mutation for Mild Hemophilia A Patients without Found DNA Mutation

Background: Identifying genetic mutations in hemophiliacs is impor-tant for disease diagnosis and genetic counseling. There are 2~7% of Hemophilia A (HA) patients whose mutations cannot be found by DNA analysis and it increases up to 10~18% in mild HA patients.Aims: We aimed to identify the genetic defects by mRNA analysis in mild-type HA patients without DNA mutation.Methods: From our 49 mild-type HA patients of 35 unrelated families from two hemophilia centers, who had undergone genetic tests from 2008 to 2016, we collected those patients without found DNA muta-tion and performed mRNA analysis. Total cellular RNA was reverse-transcribed to cDNA. F8 cDNA was amplified as eight fragments using nested PCR.
Results: There were 10 mild-type HA patients from 6 unrelated fami-lies without found mutation, which accounted for 17.1% (6/35). The median age was 18 years old, ranging from 2 to 73. The average FVIII:C level was 12.3%, ranging from 6.5% to 28.9%. Nine of the 10 (90%) patients were confirmed to have F8 gene mutation. Eight patients from 5 unrelated families were remarkably found to have both presence of normal 585 bp and aberrant 675 bp fragments. Sequencing of the aberrant 675 bp fragment showed that there were two separate insertion of 35 bp and 55 bp from intron 18. It confirmed that there was 90 bp of intron 18 insertion between exon 18 and exon 19 (E18ins90bpE19) in mRNA, which was reported as a result of c.5999-277G>A in deep intron. Another one patient was found to have exon 19 spliced out and the last one patient remained nonconclusive.Conclusions: Our study demonstrates that mRNA analysis was an effective assay for mild-type HA patients who was wild type by DNA analysis and the undetected rate can be reduced from 17.1% (6/35) to 2.9% (1/35). The E18ins90bpE19 (90-bp insertion of deep intron between E18 and E19) may be a hot spot for mild-type hemophilia without found DNA mutation.