Chia-Yau Chang1,2, Cherng-Lih Perng3,4, Shin-Nan Cheng5,6,7, Shu-Hsia Hu5, Tzu-Ying Wu5, Shyr-Yi Lin1,8, Yeu-Chin Chen5,9. 1. School of Medicine, Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan. 2. Division of Pediatric Hematology/Oncology, Hemophilia Center, Taipei Medical University Hospital, Taipei, Taiwan. 3. Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, Taipei, Taiwan. 4. Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan. 5. Hemophilia Care and Research Center, Tri-Service General Hospital, Taipei, Taiwan. 6. Department of Pediatrics, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan. 7. Department of Pediatrics, National Defense Medical Center, Taipei, Taiwan. 8. Division of Gastroenterology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan. 9. Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Abstract
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.
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 hemophiliapatients without mutation in exonic DNA.