Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

Abstract

Hemophilia A is a common X-linked bleeding disorder caused by various types of mutations in the factor VIII gene F8C. The most common intron 22-inversion is responsible for about 40% of the severe hemophilia A cases while large deletions, point mutations and small (less than 100 bp) deletions or insertions are responsible for the disease in the rest of patients. We report on nine novel (6 deletions, two indels and one partial duplication) and five recurrent small rearrangements identified in 15 German patients with severe hemophilia A, negative for the intron 22-inversion. c.2208-2214delTTATTAC/c.2207-2215insCTCTT and c.4665-4678del/c.4664-4678insAAGGAA identified in the present study are the first small indels described in the factor VIII gene. Our analyses suggest that the prevalence of this type of mutations (predominantly located in exon 14) among patients with severe phenotype and negative for the common intron 22-inversion, is about 30%. The correlation between these molecular defects and formation of factor VIII inhibitors as well as the parental origin of the de novo mutations are evaluated. Finally we show that denaturing HPLC (DHPLC) and classic heteroduplex analysis (HA) are able to detect these sequence alterations on 100% and could be preferred as a screening approach when analysing for mutations in factor VIII in severely affected patients.

Mutation analysis of factor VIII in Korean patients with severe hemophilia A

“…The previously reported recurrent mutation c.3637del A [29], a single nucleotide deletion in a 9 poly-A section of exon 14, was identified in two unrelated patients. The poly-A segment of exon 14 is considered a hotspot for mutations in hemophilia A patients [6,30].…”

Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A

“…The molecular analyses of 21 of these cases were previously published [Bogdanova et al, 2001[Bogdanova et al, , 2002Leuer et al, 2001] and the remaining 65 patients were analyzed in the present work. All of the individuals were affected by severe hemophilia A according to the standard criteria [Antonarakis et al, 1995], including one person (PL) [Bogdanova et al, 2001] who had severe hemophilia A and was initially incorrectly reported to us as being moderately affected.…”

“…All four inhibitor-positive patients (the previously published individuals NB2, NB56, and NB58 [Bogdanova et al, 2002], and NB253 (c.3385delC, exon 14)) carried small deletions in exon 14 of the F8 gene. Thus the inhibitorpositive cases represented 4.6% of our patient cohort.…”

“…To prevent ascertainment bias, we analyzed the spectrum and distribution of the mutations in all 86 severely affected individuals who were negative for the IVS22-inversion, including the previously described 21 cases [Bogdanova et al, 2001[Bogdanova et al, , 2002Leuer et al, 2001]. Supplementary Table S2 describes the frequency of all mutations identified in the present study and in our three previous works.…”

“…PCR amplification of all 26 F8 exons, including flanking intronic regions, was performed on 50-100 ng of extracted DNA using previously described amplification primers and cycling conditions [Bogdanova et al, 2002;Schwaab et al, 1997]. Amplicons were sequenced in both directions using the ABI PRISM Dye terminator cycle sequencing reaction kit (Applied Biosystems; www.appliedbiosystems.com), and electrophoresis was performed on an ABI-3700 genetic analyzer.…”