The Etiology of Hemophilia Hiding Deep Inside the F8 Intronic Sequence

Abstract 1223 Introduction/Background: Hemophilia A is a congenital X-linked bleeding disorder caused by various mutations in the coagulation factor VIII gene (F8). However, recent studies have described that no genetic mutation could be found in the F8 of about 2% of hemophilia A patients, even after nucleotide sequencing including the entire coding region, exon/intron boundaries, and the 5'- and 3'-untranslated region (Vidal et al, 2001; Klopp et al, 2002). Factor VIII deficient mechanisms underlying this phenomenon remain unexplained. To further elucidate the mechanisms causing hemophilia A in these patients, we performed a detailed analysis of F8 mRNA. Materials and methods: F8 mRNA from a Japanese hemophilia A patient with undetectable mutations was analyzed. Total RNA was isolated from peripheral blood cells using a QIAamp® RNA Blood Mini Kit (Qiagen) or PAXgene® Blood RNA Kit (Qiagen). Both preparations were performed following the manufacturer's instructions. In order to analyze the F8 mRNA, we performed the cDNA-amplification in two rounds of PCR using the nested approach reported by El-Maarri et al (2005). The nucleotide sequences of primer used followed those of their report. OneStep RT-PCR Kit (Qiagen) and TaKaRa LA Taq ™ (TaKaRa) were used for first and second round PCR amplification, respectively. Ectopic F8 mRNA expression level was relatively quantified by a real-time PCR technique using 4 TaqMan gene expression assays (Hs00240767_m1 amplify exon 1–2 boundary, Hs01109548_m1 amplify exon 6–7 boundary, Hs01109541_m1 amplify exon 14–15 boundary, Hs01109543_m1 amplify exon 20–21 boundary; Applied Biosystems). Results: Because the size of the F8 mRNA is very large ∼9kb, the entire F8 cDNA was divided into four different regions: exons 1–8 (region A); exons 8–14 (region B); exons 14–22 (region C); and exons 19–26 (region D) and amplified in the first round. Then, each of four regions were further divided into two different regions (a total of 8 overlapping regions; region 1–8), and amplified in the second round. An abnormality was observed in the amplification. Although the PCR products of regions 1 and 2, (region A), were obtained, the products remaining in all later regions (regions 3–8) were not. A similar phenomenon was also confirmed in the semi-quantification of the mRNA. Though we were able to quantify the mRNA by using both exon 1–2 and 5–6 boundary amplifications, we were not able to quantify the mRNA using the 14–15 and 20–21 boundaries. These results suggested that the quantity of the mRNA decrease remarkably in the vicinity of exon 8 as a boundary. Further analysis of the mRNA showed that quantity of the mRNA is normal from exon 1 through 9. Nucleotide sequencing of intron 9 revealed a single nucleotide substitution, adenine to guanine transition, at 602bp downstream from the 3' end of exon 9. This transition has not been registered in any international database as a mutation or a polymorphism and was not found in the F8 from 124 Japanese. These results strongly suggest that the transition is very rare and may be involved in factor VIII deficiency in these patients. Analysis of the nucleotide sequence of the substitution by splicing site prediction software predicted the formation of a new acceptor splice site. This result suggested the existence of splice abnormality. However, further characterization is needed to elucidate the mechanism that causes the decrease in mRNA in the middle of the gene. Conclusion: The mechanism behind factor VIII deficiency in hemophilia A patients with undetectable mutations is very interesting and various possibilities are conceivable. This study provides the possibility that some causative genetic abnormality remains in a further unanalyzed F8 region, most likely deep inside the intron, of these patients. Disclosures: No relevant conflicts of interest to declare.