RSS-Feed abonnieren
DOI: 10.1055/s-0028-1103360
Molecular Genetic Testing of Hemophilia A
Publikationsverlauf
Publikationsdatum:
28. November 2008 (online)
![](https://www.thieme-connect.de/media/sth/200806/lookinside/thumbnails/10.1055-s-0028-1103360-1.jpg)
ABSTRACT
Genetic testing in hemophilia A continues to diversify. This article describes recent advances in several aspects of genetic analysis and its interpretation and reporting. The intron 1 and 22 inversions responsible for 50% of severe hemophilia A cases can be sought using long and inverse polymerase chain reaction (PCR) techniques. Point mutations are analyzed in remaining patients by PCR amplification of the F8 protein-coding region followed by either mutation screening to identify the mutated amplicon and subsequent DNA sequencing or by directly sequencing amplified DNA. Many technique modifications and sequence analysis software packages are available to reduce time and effort required to identify a mutation. Dosage analysis and gap PCR have been described to identify carriers of large F8 deletions. Noninvasive prenatal fetal sex determination and preimplantation genetic diagnosis extend the reproductive options available to hemophilia carriers. Guidelines on undertaking and reporting the testing plus external quality assessment are now available to help ensure that genetic analysis yields accurate and well-interpreted results.
KEYWORDS
Hemophilia A - mutation - molecular pathology - genetic analysis
REFERENCES
- 1 HAMSTeRS .The Haemophilia A Mutation, Structure, Test and Resource Site. Available at http://europium.csc.mrc.ac.uk/WebPages/Main/main.htm Accessed January 10, 2008
- 2 HGMD .The Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff. Available at http://www.hgmd.cf.ac.uk/ac/index.php Accessed January 10, 2008
- 3 Lakich D, Kazazian Jr H H, Antonarakis S E, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet. 1993; 5 236-241
- 4 Bagnall R D, Waseem N, Green P M, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood. 2002; 99 168-174
- 5 Ghosh K, Shetty S, Mohanty D. Inversion of intron 1 is a rare cause of severe hemophilia A in Indian population. J Thromb Haemost. 2004; 2 1481-1482
- 6 Cumming A M. The factor VIII gene intron 1 inversion mutation: prevalence in severe hemophilia A patients in the UK. J Thromb Haemost. 2004; 2 205-206
- 7 Muhle C, Zenker M, Chuzhanova N, Schneider H. Recurrent inversion with concomitant deletion and insertion events in the coagulation factor VIII gene suggests a new mechanism for X-chromosomal rearrangements causing hemophilia A. Hum Mutat. 2007; 28 1045
- 8 Bogdanova N, Markoff A, Pollmann H et al.. Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A. Hum Mutat. 2005; 26 249-254
- 9 Santacroce R, Acquila M, Belvini D et al.. Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A. J Hum Genet. 2008; 53 275-284
- 10 Vidal F, Farssac E, Tusell J, Puig L, Gallardo D. First molecular characterization of an unequal homologous alu-mediated recombination event responsible for hemophilia. Thromb Haemost. 2002; 88 12-16
- 11 Rossetti L C, Goodeve A, Larripa I B, De Brasi C D. Homeologous recombination between AluSx-sequences as a cause of hemophilia. Hum Mutat. 2004; 24 440
- 12 Nakaya S M, Hsu T C, Geraghty S J, Manco-Johnson M J, Thompson A R. Severe hemophilia A due to a 1.3 kb factor VIII gene deletion including exon 24: homologous recombination between 41 bp within an Alu repeat sequence in introns 23 and 24. J Thromb Haemost. 2004; 2 1941-1945
- 13 Van de Water N, Williams R, Ockelford P, Browett P. A 20.7 kb deletion within the factor VIII gene associated with LINE-1 element insertion. Thromb Haemost. 1998; 79 938-942
- 14 Kazazian Jr H H, Wong C, Youssoufian H, Scott A F, Phillips D G, Antonarakis S E. Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature. 1988; 332 164-166
- 15 Ganguly A, Dunbar T, Chen P, Godmilow L, Ganguly T. Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A. Hum Genet. 2003; 113 348-352
- 16 Sukarova E, Dimovski A J, Tchacarova P, Petkov G H, Efremov G D. An Alu insert as the cause of a severe form of hemophilia A. Acta Haematol. 2001; 106 126-129
- 17 Sheen C R, Jewell U R, Morris C M et al.. Double complex mutations involving F8 and FUNDC2 caused by distinct break-induced replication. Hum Mutat. 2007; 28 1198-1206
- 18 Bogdanova N, Markoff A, Pollmann H et al.. Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A. Hum Mutat. 2002; 20 236-237
- 19 Dai L, Cutler J A, Savidge G F, Mitchell M J. Characterization of a causative mutation of hemophilia A identified in the promoter region of the factor VIII gene (F8). J Thromb Haemost. 2008; 6 193-195
- 20 Bogdanova N, Markoff A, Eisert R et al.. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat. 2007; 28 54-60
- 21 Acquila M, Pasino M, Lanza T, Bottini F, Molinari A C, Bicocchi M P. Duplication of exon 13 causes one third of the cases of mild hemophilia A in northern Italy. Haematologica. 2004; 89 758-759
- 22 Oldenburg J, Ivaskevicius V, Rost S et al.. Evaluation of DHPLC in the analysis of hemophilia A. J Biochem Biophys Methods. 2001; 47 39-51
- 23 Vinciguerra C, Zawadzki C, Dargaud Y et al.. Characterization of 96 mutations in 128 unrelated severe haemophilia A patients from France. Description of 62 novel mutations. Thromb Haemost. 2006; 95 593-599
- 24 Jayandharan G, Shaji R V, Baidya S, Nair S C, Chandy M, Srivastava A. Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions. Haemophilia. 2005; 11 481-491
- 25 Bagnall R D, Waseem N H, Green P M, Colvin B, Lee C, Giannelli F. Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients. Br J Haematol. 1999; 107 766-771
- 26 El-Maarri O, Herbiniaux U, Graw J et al.. Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene. J Thromb Haemost. 2005; 3 332-339
- 27 El-Maarri O, Singer H, Klein C et al.. Lack of F8 mRNA: a novel mechanism leading to hemophilia A. Blood. 2006; 107 2759-2765
- 28 Mazurier C, Hilbert L. Type 2N von Willebrand disease. Curr Hematol Rep. 2005; 4 350-358
- 29 Schneppenheim R, Budde U, Krey S et al.. Results of a screening for von Willebrand disease type 2N in patients with suspected haemophilia A or von Willebrand disease type 1. Thromb Haemost. 1996; 76 598-602
- 30 Nesbitt I M, Goodeve A C, Guilliatt A M, Makris M, Preston F E, Peake I R. Characterisation of type 2N von Willebrand disease using phenotypic and molecular techniques. Thromb Haemost. 1996; 75 959-964
- 31 ISTH-VWF-SSC . International Society on Thrombosis and Haemostasis Scientific and Standardization Committee VWF Information Homepage. Available at http://www.vwf.group.shef.ac.uk/ Accessed March 23, 2008;
- 32 Zhang B, Ginsburg D. Familial multiple coagulation factor deficiencies: new biologic insight from rare genetic bleeding disorders. J Thromb Haemost. 2004; 2 1564-1572
- 33 Zhang B, McGee B, Yamaoka J S et al.. Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2. Blood. 2006; 107 1903-1907
- 34 Liu Q, Nozari G, Sommer S S. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Blood. 1998; 92 1458-1459
- 35 Liu Q, Sommer S S. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene. Biotechniques. 1998; 25 1022-1028
- 36 Kilian N L, Pospisil V, Hanrahan V. Haemophilia A, factor VIII intron 22 inversion screening using subcycling-PCR. Thromb Haemost. 2006; 95 746-747
- 37 Vidal F, Sanchez-Garcia J F, Farssac E, Ramirez L, Gallardo D. Rapid single-step detection of the inversion hotspot of mutation in hemophilia A by real-time PCR. J Thromb Haemost. 2005; 3 2822-2823
- 38 Bagnall R D, Giannelli F, Green P M. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost. 2006; 4 591-598
- 39 Rossetti L C, Radic C P, Larripa I B, De Brasi C D. Genotyping the hemophilia inversion hotspot by use of inverse PCR. Clin Chem. 2005; 51 1154-1158
- 40 Bowen D J, Keeney S. Unleashing the long-distance PCR for detection of the intron 22 inversion of the factor VIII gene in severe haemophilia A. Thromb Haemost. 2003; 89 201-202
- 41 Rossetti L C, Radic C P, Larripa I B, De Brasi C D. Developing a new generation of tests for genotyping hemophilia causative rearrangements involving int22h and int1h hotspots in the factor 8 gene. J Thromb Haemost. 2008; 6 830-836
- 42 Habart D, Kalabova D, Novotny M, Vorlova Z. Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions. J Thromb Haemost. 2003; 1 773-781
- 43 Repesse Y, Slaoui M, Ferrandiz D et al.. Factor VIII (FVIII) gene mutations in 120 patients with hemophilia A: detection of 26 novel mutations and correlation with FVIII inhibitor development. J Thromb Haemost. 2007; 5 1469-1476
- 44 Williams I J, Abuzenadah A, Winship P R et al.. Precise carrier diagnosis in families with haemophilia A: use of conformation sensitive gel electrophoresis for mutation screening and polymorphism analysis. Thromb Haemost. 1998; 79 723-726
- 45 Goodeve A C, Williams I, Bray G L, Peake I R. Relationship between factor VIII mutation type and inhibitor development in a cohort of previously untreated patients treated with recombinant factor VIII (Recombinate). Recombinate PUP Study Group. Thromb Haemost. 2000; 83 844-848
- 46 Davies H, Dicks E, Stephens P et al.. High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison. Genomics. 2006; 87 427-432
- 47 Laurie A D, Smith M P, George P M. Detection of factor VIII gene mutations by high-resolution melting analysis. Clin Chem. 2007; 53 2211-2214
- 48 Keeney S, Watson P, Hay C, Cumming A. Rapid turnaround for full mutation analysis of larger genes: robotic processing and automated DNA sequencing of the essential regions of the FVIII (F8) gene for mutation identification in haemophilia A. J Med Genet. 2004; 41(Suppl1) S67
- 49 Vidal F, Farssac E, Altisent C, Puig L, Gallardo D. Rapid hemophilia A molecular diagnosis by a simple DNA sequencing procedure: identification of 14 novel mutations. Thromb Haemost. 2001; 85 580-583
- 50 Staden Package homepage. Available at http://staden.sourceforge.net/ Accessed March 3, 2008
- 51 Akkarapatumwong V, Oranwiroon S, Pung-amritt P et al.. Mutations of the factor VIII gene in Thai hemophilia A patients. Hum Mutat. 2000; 15 117-118
- 52 Waseem N H, Bagnall R, Green P M, Giannelli F. Start of UK confidential haemophilia A database: analysis of 142 patients by solid phase fluorescent chemical cleavage of mismatch. Haemophilia Centres. Thromb Haemost. 1999; 81 900-905
- 53 Rainen L, Oelmueller U, Jurgensen S et al.. Stabilization of mRNA expression in whole blood samples. Clin Chem. 2002; 48 1883-1890
- 54 Fernandez-Lopez O, Garcia-Lozano J R, Nunez-Vazquez R, Perez-Garrido R, Nunez-Roldan A. Characterization of sequence breakpoints in two haemophiliac patients with large FVIII gene deletions. Haemophilia. 2007; 13 682-684
- 55 Pavlova A, Forster T, Delev D et al.. Heterozygous large deletions of factor 8 gene in females identified by multiplex PCR-LC. Haemophilia. 2008; 14 599-606
- 56 Tizzano E F, Barcelo M J, Baena M et al.. Rapid identification of female haemophilia A carriers with deletions in the factor VIII gene by quantitative real-time PCR analysis. Thromb Haemost. 2005; 94 661-664
- 57 MRC-Holland .MRC-Holland MLPA homepage. Available at http://www.mlpa.com/pages/indexpag.html Accessed February 26, 2008
- 58 Lannoy N, Abinet I, Verellen C, Vermylen C, Hermans K, Dahan K. Proposition for a multi-step mutation detection in haemophilia A. ISTH 2007 Congress abstracts 2007:ISTH07A01_P-M-138
- 59 Lin H Y, Chen M, Chang H H et al.. Severe haemophilia A and factor VIII inhibitor occurs in the young girl who has a carrier mother, Turner's syndrome and chromosomal structural abnormalities with 45X(22)/46,X,IDIC(X)(Q21)(8). ISTH 2007 Congress abstracts 2007; P-M-142
- 60 Djambas Khayat C, Salem N, Chouery E et al.. Detection of 23 novel mutations in Lebanese hemophilia patients. ISTH 2007 Congress abstracts 2007; P-S-156
- 61 Jayandharan G, Shaji R V, George B, Chandy M, Srivastava A. Informativeness of linkage analysis for genetic diagnosis of haemophilia A in India. Haemophilia. 2004; 10 553-559
- 62 Soares R P, Chamone D A, Bydlowski S P. Factor VIII gene inversions and polymorphisms in Brazilian patients with haemophilia A: carrier detection and prenatal diagnosis. Haemophilia. 2001; 7 299-305
- 63 Azimifar S B, Seyedna S Y, Zeinali S. Allele frequencies of three factor VIII gene polymorphisms in Iranian populations and their application in hemophilia A carrier detection. Am J Hematol. 2006; 81 335-339
- 64 Viel K R, Machiah D K, Warren D M et al.. A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels. Blood. 2007; 109 3713-3724
- 65 NCBI db SNP webpage. Available at http://www.ncbi.nlm.nih.gov/projects/SNP/ Accessed February 26, 2008
- 66 Kim J W, Park S Y, Kim Y M, Kim J M, Kim D J, Ryu H M. Identification of new dinucleotide-repeat polymorphisms in factor VIII gene using fluorescent PCR. Haemophilia. 2005; 11 38-42
- 67 Harraway J R, Smith M P, George P M. A highly informative, multiplexed assay for the indirect detection of hemophilia A using five-linked microsatellites. J Thromb Haemost. 2006; 4 587-590
- 68 Chowdhury M R, Mathur R, Verma I C. Utility of XY-amelogenin gene primers for detection of sex chromosomes. Indian J Med Res. 1998; 107 182-186
- 69 Schrijver I, Cherny S C, Zehnder J L. Testing for maternal cell contamination in prenatal samples: a comprehensive survey of current diagnostic practices in 35 molecular diagnostic laboratories. J Mol Diagn. 2007; 9 394-400
- 70 Santacroce R, Vecchione G, Tomaiyolo M et al.. Identification of fetal gender in maternal blood is a helpful tool in the prenatal diagnosis of haemophilia. Haemophilia. 2006; 12 417-422
- 71 Chi C, Hyett J A, Finning K M, Lee C A, Kadir R A. Non-invasive first trimester determination of fetal gender: a new approach for prenatal diagnosis of haemophilia. BJOG. 2006; 113 239-242
- 72 Sekizawa A, Purwosunu Y, Matsuoka R et al.. Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood. J Obstet Gynaecol Res. 2007; 33 747-764
- 73 Michaelides K, Tuddenham E G, Turner C, Lavender B, Lavery S A. Live birth following the first mutation specific pre-implantation genetic diagnosis for haemophilia A. Thromb Haemost. 2006; 95 373-379
- 74 Lavery S. Preimplantation genetic diagnosis: new reproductive options for carriers of haemophilia. Haemophilia. 2004; 10(Suppl 4) 126-132
- 75 Renwick P, Ogilvie C M. Preimplantation genetic diagnosis for monogenic diseases: overview and emerging issues. Expert Rev Mol Diagn. 2007; 7 33-43
- 76 Human Genome Variation Society .Nomenclature for the description of sequence variations webpage. Available at http://www.hgvs.org/mutnomen/ Accessed March 2, 2008
- 77 Keeney S, Mitchell M, Goodeve A. The molecular analysis of haemophilia A: a guideline from the UK haemophilia centre doctors' organization haemophilia genetics laboratory network. Haemophilia. 2005; 11 387-397
- 78 Bell J, Bodmer D, Sistermans E, Ramsden S. Practice Guidelines for the Interpretation and Reporting of Unclassified Variants (UVs) in Clinical Molecular Genetics. Available at http://cmgsweb.shared.hosting.zen.co.uk/BPGs/Best_Practice_Guidelines.htm Accessed March 2, 2008
- 79 Perry D J, Goodeve A, Hill M, Jennings I, Kitchen S, Walker I. The UK National External Quality Assessment Scheme (UK NEQAS) for molecular genetic testing in haemophilia. Thromb Haemost. 2006; 96 597-601
- 80 Castaman G, Giacomelli S H, Ghiotto R et al.. Spectrum of mutations in Albanian patients with haemophilia A: identification of ten novel mutations in the factor VIII gene. Haemophilia. 2007; 13 311-316
- 81 Boekhorst J, Verbruggen B, Lavergne J M et al.. Thirteen novel mutations in the factor VIII gene in the Nijmegen haemophilia A patient population. Br J Haematol. 2005; 131 109-117
- 82 Guillet B, Lambert T, d'Oiron R et al.. Detection of 95 novel mutations in coagulation factor VIII gene F8 responsible for hemophilia A: results from a single institution. Hum Mutat. 2006; 27 676-685
Anne GoodevePh.D.
Honorary Clinical Scientist and Reader in Molecular Medicine, Sheffield Molecular Genetics Service
Sheffield Children's NHS Foundation Trust, Western Bank, Sheffield S10 2TH, UK
eMail: a.goodeve@shef.ac.uk