Lack of F8 mRNA: a novel mechanism leading to hemophilia A

El‐Maarri, Osman; Singer, Heike; Klein, Claudia; Watzka, Matthias; Herbiniaux, Ursula; Brackmann, H.‐H.; Schröder, Jörg; Graw, Jochen; Müller, C. R.; Schramm, W.; Schwaab, R.; Haaf, Thomas; Hanfland, P.; Oldenburg, Johannes

doi:10.1182/blood-2005-09-3702

Cited by 26 publications

(29 citation statements)

References 23 publications

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“…This has been reported by several workers earlier, wherein mutations have not been detected in 2–10% of the HA cases, even after directly sequencing the coding, splice site and the promoter region of F8 [30], [31]. Deep intronic mutations, epigenetic factors are some of the possible explanations which need further evaluation.…”

Section: Discussionmentioning

confidence: 50%

Mutations in Intron 1 and Intron 22 Inversion Negative Haemophilia A Patients from Western India

et al. 2014

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Despite increased awareness and diagnostic facilities, 70–80% of the haemophilia A (HA) patients still remain undiagnosed in India. Very little data is available on prevalent mutations in HA from this country. We report fifty mutations in seventy one Indian HA patients, of which twenty were novel. Ten novel missense mutations [p.Leu11Pro (p.Leu-8Pro), p.Tyr155Ser (p.Tyr136Ser), p.Ile405Thr (p.Ile386Thr), p.Gly582Val (p.Gly563Val) p.Thr696Ile (p.Thr677Ile), p.Tyr737Cys (p.Tyr718Cys), p.Pro1999Arg (p.Pro1980Arg), p.Ser2082Thr (p.Ser2063Thr), p.Leu2197Trp (p.Leu2178Trp), p.Asp2317Glu (p.Asp2298Glu)] two nonsense [p.Lys396* (p.Lys377*), p.Ser2205* (p.Ser2186*)], one insertion [p.Glu1268_Asp1269ins (p.Glu1249_Asp1250)] and seven deletions [p.Leu882del (p.Leu863del), p.Met701del (p.Met682del), p.Leu1223del (p.Leu1204del), p.Trp1961_Tyr1962del (p.Trp1942_Tyr1943del) p.Glu1988del (p.Glu1969del), p.His1841del (p.His1822del), p.Ser2205del (p.Ser2186del)] were identified. Double mutations (p.Asp2317Glu; p.Thr696Ile) were observed in a moderate HA case. Mutations [p. Arg612Cys (p.Arg593Cys), p.Arg2326Gln (p.Arg2307Gln)] known to be predisposing to inhibitors to factor VIII (FVIII) were identified in two patients. 4.6% of the cases were found to be cross reacting material positive (CRM+ve). A wide heterogeneity in the nature of mutations was seen in the present study which has been successfully used for carrier detection and antenatal diagnosis in 10 families affected with severe to moderate HA.

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Section: Discussionmentioning

confidence: 50%

Mutations in Intron 1 and Intron 22 Inversion Negative Haemophilia A Patients from Western India

et al. 2014

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“…Therefore there is no assurance that the effect of the variations found here will be the same in FVIII expressing/secreting cells. Nevertheless, the importance of screening for splicing variations has been underlined in HA and our work highlights the importance of implementation of F8 mRNA analysis in clinical practice.…”

Section: Discussionmentioning

confidence: 86%

Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA

Pezeshkpoor

Zimmer

Marquardt

et al. 2013

Journal of Thrombosis and Haemostasis

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Summary. Background: In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and nongenetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. Methods: We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LRPCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. Results: All VWF tests were normal. The LRPCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. Conclusion: By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

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“…This rearrangement leads to the second event wherein an intra‐chromosomal homologous recombination occurs between F8 :Int1R‐1 and Int1R‐2d in the duplicated IKBKG gene. As a result, F8 is split in intron 1 into two oppositely oriented fragments for which no functional mRNA is transcribed [8]. On the other hand, the essential IKBKG gene is still intact and transcribed, as the homologous recombination involves the duplicated copy and not the endogenous IKBKG or IKBKGP .…”

Section: Discussionmentioning

confidence: 99%

“…The index patient is a male hemophilia A patient with a severe phenotype (< 1 IU dL −1 factor [F]VIII activity) who suffers from no other known health problems as previously reported [8]. No inhibitors have been detected.…”

Section: Methodsmentioning

confidence: 99%

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Identification of a third rearrangement at Xq28 that causes severe hemophilia A as a result of homologous recombination between inverted repeats

Pezeshkpoor

Rost

Oldenburg

et al. 2012

Journal of Thrombosis and Haemostasis

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To cite this article: Pezeshkpoor B, Rost S, Oldenburg J, El-Maarri O. Identification of a third rearrangement at Xq28 that causes severe hemophilia A as a result of homologous recombination between inverted repeats. J Thromb Haemost 2012; 10: 1600-8.Summary. Background: Intrachromosomal homologous recombination between inverted repeats on the X chromosome account for about half of severe hemophilia A cases. Repeats in F8 intron 1 and intron 22 can recombine with homologous inverted repeats located about 200 kb upstream and 500 kb downstream of F8, respectively, resulting in partial sequence inversion of the F8 open reading frame and, subsequently, no functional protein production. Objectives: In the present study, we characterize a third novel homologous recombination at Xq28 consistent with absence of F8 transcription that we previously reported for the affected chromosome of the index patient as well as his mother and sister. Results: The rearrangement occurs between a repeat in F8 intron 1 (Int1R-1) and an inverted identical repeat (Int1R-2d) in intron 2 of a duplicated copy of IKBKG located about 386 kb upstream of F8. The rearrangement was confirmed by Southern blot and inverse PCR and results in failure of PCR amplification across Int1R-1. Conclusion: We developed a PCR-based diagnostic method that can be used to screen for this genetic rearrangement in cases of severe hemophilia A for which mutations cannot be identified.

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Lack of F8 mRNA: a novel mechanism leading to hemophilia A

Cited by 26 publications

References 23 publications

Mutations in Intron 1 and Intron 22 Inversion Negative Haemophilia A Patients from Western India

Mutations in Intron 1 and Intron 22 Inversion Negative Haemophilia A Patients from Western India

Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA

Identification of a third rearrangement at Xq28 that causes severe hemophilia A as a result of homologous recombination between inverted repeats

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