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DOI: 10.1160/TH16-05-0375
Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes
Publikationsverlauf
Received:
15. Mai 2016
Accepted after major revision:
17. September 2016
Publikationsdatum:
01. Dezember 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/201701/lookinside/thumbnails/10-1160-th16-05-0375-1.jpg)
Summary
Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.
Supplementary Material to this article is available online at www.thrombosis-online.com.
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References
- 1 Blanchette VS, Key NS, Ljung LR. Subcommittee on Factor VIII, Factor IX and Rare Coagulation Disorders. Definitions in haemophilia: communication from the SSC of the ISTH. J Thromb Haemost 2014; 12: 1935-1939.
- 2 Bolton-Maggs PH, Pasi KJ. Haemophilias A and B. Lancet 2003; 361: 1801-1809.
- 3 Mårtensson A, Ivarsson S, Letelier A. et al. Origin of mutation in sporadic cases of severe haemophilia A in Sweden. Clin Genet 2016; 90: 63-68.
- 4 Ranjan R, Biswas A, Meena A. et al. Importance of investigating somatic and germline mutations in haemophilia A: a preliminary study from All India Institute of Medical Sciences, India. Clin Chim Acta 2008; 389: 103-108.
- 5 Coppola A, Margaglione M, Santagostino E. et al. AICE PROFIT Study Group. Factor VIII gene (F8) mutations as predictors of outcome in immune tolerance induction of haemophilia A patients with high-responding inhibitors. J Thromb Haemost 2009; 07: 1809-1815.
- 6 Oldenburg J, Ananyeva NM, Saenko EL. Molecular basis of haemophilia A. Haemophilia 2004; 10: 133-139.
- 7 Rosset C, Vieira IA, Sinigaglia M. et al. Detection of new mutations and molecular pathology of mild and moderate haemophilia A patients from southern Brazil. Haemophilia 2013; 19: 773-781.
- 8 Bastida JM, Del Rey M, Lozano ML. et al. Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders. Haemophilia 2016; 22: 590-597.
- 9 Bach JE, Wolf B, Oldenburg J. et al. Identification of deep intronic variants in 15 haemophilia A patients by next generation sequencing of the whole factor VIII gene. Thromb Haemost 2015; 114: 757-767.
- 10 Boylan B, Rice AS, De Staercke C. et al. Haemophilia Inhibitor Research Study Investigators. Evaluation of von Willebrand factor phenotypes and genotypes in Haemophilia A patients with and without identified F8 mutations. J Thromb Haemost 2015; 13: 1036-1042.
- 11 Liu Q, Sommer SS. Subcycling-PCR for multiplex long distance amplification of regions with high and low GC content: application to the inversion hotspot in the FVIII gene. Biotechniques 1998; 25: 1022-1028.
- 12 Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe haemophilia A. Blood 2002; 99: 168-174.
- 13 Keeney S, Mitchell M, Goodeve A. The molecular analysis of haemophilia A: a guideline from the UK haemophilia centre doctors´ organisation haemophilia genetics laboratory network. Haemophilia 2005; 11: 387-397.
- 14 Lannoy N, Abinet I, Dahan K, Hermans C. Identification of de novo deletion in the factor VIII gene by MLPA technique in two girls with isolated factor VIII deficiency. Haemophilia 2009; 15: 797-801.
- 15 Santacroce R, Longo V, Bafunno V. et al. Detection of new deletions in a group of Italian patients with Haemophilia A by multiplex ligation-dependent probe amplification. Genet Test Mol Biomarkers 2009; 13: 573-576.
- 16 Andrews RM, Kubacka I, Chinnery PF. et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999; 23: 147.
- 17 Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 2010; 26: 589-595.
- 18 Pandey RV, Pabinger S, Kriegner A. et al. Sanger and NGS based integrated pipeline for mutation identification, validation and annotation in human molecular genetics. PLoS One 2016; 11: e0147697.
- 19 DePristo MA, Banks E, Poplin R. et al. A framework for variation discovery and genotyping using Next-Generation DNA sequencing data. Nat Genet 2011; 43: 491-498.
- 20 Richards S, Aziz N, Bale S. et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17: 405-424.
- 21 Jian X, Boerwinkle E, Liu X. In silico prediction of splice-altering single nucleotide variants in the human genome. Nucleic Acids Res 2014; 42: 13534-13544.
- 22 Ionita-Laza I, McCallum K, Xu B, Buxbaum JD. A spectral approach integrating functional genomic annotations for coding and noncoding variants. Nat Genet 2016; 48: 214-220.
- 23 Keeney S. Steve Keeney’s Primers for PCR amplification. HAMSTeRS. The Haemophilia A Mutation, Structure, Test and Resource Site Available from: http://hadb.org.uk/WebPages/Database/Methods/pcr.html
- 24 Vidal F, Farssac E, Altisent C. et al. Factor IX gene sequencing by a simple and sensitive 15-hour procedure for haemophilia B diagnosis: identification of two novel mutations. Br J Haematol 2000; 111: 549-551.
- 25 Corrales I, Ramirez L, Altisent C. et al. Rapid molecular diagnosis of von Willebrand disease by direct sequencing. Detection of 12 novel putative mutations in VWF gene. Thromb Haemost 2009; 101: 570-576.
- 26 Lu JT, Campeau PM, Lee BH. Genotype-phenotype correlation promiscuity in the era of next-generation sequencing. New Engl J Med 2014; 371: 593-596.
- 27 Sikkema-Raddatz B, Johansson LF, de Boer EN. et al. Targeted Next-Generation sequencing can replace Sanger sequencing in clinical diagnostics. Hum Mutat 2013; 34: 1035-1042.
- 28 Batlle J, Pérez-Rodríguez A, Corrales I. et al. Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): Proposal for a new diagnostic paradigm. Thromb Haemost 2015; 115: 40-50.
- 29 Mackie I, Cooper P, Lawrie A. et al. British Committee for Standards in Haematology. Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis. Int J Lab Hematol 2013; 35: 1-13.
- 30 Bardi E, Astermark J. Genetic risk factors for inhibitors in haemophilia A. Eur J Haematol 2015; 94: 7-10.
- 31 Santagostino E, Gringeri A, Tagliavacca L, Mannucci PM. Inhibitors to factor VIII in a family with mild haemophilia: molecular characterisation and response to factor VIII and desmopressin. Thromb Haemost 1995; 74: 619-621.
- 32 Gupta M, Lillicrap D, Stain AM. et al. Therapeutic consequences for misdiagnosis of type 2N von Willebrand disease. Pediatr Blood Cancer 2011; 57: 1081-1083.
- 33 Mazurier C, Goudemand J, Hilbert L. et al. Type 2N von Willebrand disease: clinical manifestations, pathophysiology, laboratory diagnosis, and molecular biology. Best Pract Res Clin Hematol 2001; 14: 337-347.
- 34 Feng J, Liu Q, Drost J, Sommer SS. Deep intronic mutations are rarely a cause of haemophilia B. Hum Mutat 1999; 14: 267-268.
- 35 Weaver JM, Edwards PA. Targeted Next-Generation sequencing for routine clinical screening of mutations. Genome Med 2011; 03: 58.
- 36 Kingsmore SF, Saunders CJ. Deep sequencing of patient genomes for disease diagnosis: when will it become routine?. Sci Transl Med 2011; 03: 87ps23.
- 37 Bowyer AE, Van Veen JJ, Goodeve AC. et al. Specific and global coagulation assays in the diagnosis of discrepant mild haemophilia A. Haematologica 2013; 98: 1980-1987.
- 38 Daber R, Sukhadia S, Morrissette JJ. Understanding the limitations of Next-Generation sequencing informatics, an approach to clinical pipeline validation using artificial data sets. Cancer Genet 2013; 206: 441-448.