Fast and Efficient Mutation Detection Method Using Multiplex PCR and Cycle Sequencing

Summary

A method using multiplex PCR followed by cycle-sequencing has been developed to detect mutations in the FIX gene. The procedure was evaluated in 45 severe or mild haemophilia B patients from 45 unrelated families. At least one deleterious mutation was identified in every haemophiliac demonstrating the efficiency of the method. Furthermore the described procedure offers many advantages compared to other screening detection methods: it is fast (less than 48 h), simple (partly automated) and of relatively low cost (it requires only one PCR).

Keywords

Haemophilia B - FIX gene - mutation screening

• References

• 1 Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K. Nucleotide sequence of the gene for Human Factor IX (Antihemophilic Factor B). Biochemistry 1985; 24: 3736-50.
• 2 Giannelli F, Green PM, Sommer SS, Poon MC, Ludwig M, Schwaab R, Reitsma PH, Goossens M, Yoshioka A, Brownlee GG. Haemophilia B: Database of point mutations and short additions and deletions. Nucleic Acids Res 1996; 24: 103-18.
• 3 Nielsen LR, Scheibel E, Ingerslev J, Schwartz M. Detection of ten new mutations by screening the gene encoding factor IX of Danish hemophilia B patients. Thromb Haemost 1995; 73: 774-8.
• 4 Montejo JM, Magallon M, Tizzano E, Solera J. Identification of twenty-one new mutations in the factor IX gene by SSCP analysis. Hum Mutat 1999; 13: 160-5.
• 5 Tartary M, Vidaud D, Piao Y, Costa JM, Bahnak BR, Fressinaud E, Congard B, Laurian Y, Meyer D, Lavergne JM, Vidaud M. Detection of a molecular defect in 40 of 44 patients with haemophilia B by PCR and denaturing gradient gel electrophoresis. Br J Haematol 1993; 84: 662-9.
• 6 Ghanem N, Costes B, Martin J, Vidaud M, Rothschild C, Foyer-Gazengel C, Goossens M. Twenty-four novel hemophilia B mutations revealed by rapid scanning of the whole factor IX gene in a French population sample. Eur J Hum Genet 1993; 01: 144-55.
• 7 Driscoll MC, Chu A, Hilgartner MW. Heteroduplex analysis in hemophilia B: detection of two novel factor IX gene mutations. Am J Hematol 1996; 51: 324-7.
• 8 Haris II, Green PM, Bentley DR, Giannelli F. Mutation detection by fluorescent chemical cleavage: application to hemophilia B. PCR Methods Apll 1994; 03: 268-71.
• 9 Schwaab R, Brackmann HH, Meyer C, Seehafer J, Kirchgesser M, Haack A, Olek K, Tuddenham EGD, Oldenburg J. Haemophilia A: mutation type determines risk of inhibitor formation. Thromb Haemost 1995; 74: 1402-6.
• 10 O’Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoorn B, Guy C, Speight G, Upadhyaya M, Sommer SS, McGuffin P. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 1998; 15; 52: 44-9.
• 11 Bottema CD, Koeberl DD, Sommer SS. Direct carrier testing in 14 families with haemophilia B. Lancet 1989; 256.
• 12 Goodeve AC. Advances in carrier detection in haemophilia. Haemophilia 1998; 04: 358-64.
• 13 Eggerding FA, Iovannisci DM, Brinson E, Grossman P, Winn-Deen ES. Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations. Hum Mutat 1995; 05: 153-65.
• 14 Higuchi M, Wong C, Kochhan L, Olek K, Aronis S, Kasper CK, Kazazian HH, Antonorakis SE. Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA. Genomics 1990; 06: 67-71.
• 15 Pieneman WC, Deutz-Terlouw PP, Reitsma PH, Briet E. Screening for mutations in haemophilia A patients by multiplex PCR-SSCP, Southern blotting and RNA analysis: the detection of a genetic abnormality in the FVIII gene in 30 out of 35 patients. Br J Haematol 1995; 90: 442-9.