Double Strand Conformation Polymorphism (DSCP) Detects Two Point Mutations at Codon 280 (AAC→ATC) and at Codon 431 (TAC→AAC) of the Blood Coagulation Factor VIII Gene

Summary

Hemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC→AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A→T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP).

At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.

• References

• 1 Higuchi M, Kochhan L, Schwaab R, Egli H, Brackman HH, Horst J, Olek K. Molecular defects in Hemophilia A: Identification and characterization of mutations in the factor VIII gene and family analysis. Blood 1989; 74: 1045-1051
• 2 Higuchi M, Kazazian HH, Kasch L, Warren TC, McGinniss MJ, Phillips III JA, Kasper C, Janco R, Antonarakis SE. Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene. Proc Natl Acad Sci USA 1991; 88: 7405-7409
• 3 Higuchi M, Antonarakis SE, Kasch L, Oldenburg J, Economou-Petersen E, Olek K, Arai M, Inaba H, Kazazian HH. Molecular characterization of mild-to-moderate hemophilia A: Detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1991; 88: 8307-8311
• 4 Plieth J, Rininsland F, Schlössner M, Cooper DN, Reiss J. Single strand conformation polymorphism (SSCP) analysis of exon 11 of the CFTR gene reliably detects more than one third of non-ΔF508 mutations in German cystic fibrosos patients. Hum Genet 1992; 88: 283-287
• 5 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Highichi R, Hom GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-491
• 6 Orita M, lwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 1989; 86: 2766-2770
• 7 Reitsma PH, Mandalaki F, Kasper CK, Bertina RM, Briet E. Two novel point mutations correlate with an altered developmental expression of blood coagulation factor IX (hemophilia B Leyden phenotype). 1989; Blood: 743-746
• 8 Wood WI, Capon DJ, Simonsen CC, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EGD, Vehar GA, Lawn RM. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-337
• 9 Dickerson RE, Drew HR. Structure of a B-DNA dodecamerII. Influence of base sequence on helix structure. J Mol Biol 1981; 149: 761-786
• 10 Shapiro BA, Nussinov R, Lipkin LE, Maizel Jr JV. A sequence analysis system encompassing rules for DNA helical distortion. Nucl Acid Res 1986; 14: 75-86
• 11 Marini JC, Levene SD, Crothers DM, Englund PT. Bent helical structure in kinetoplast DNA. Proc Natl Acad Sci USA 1982; 79: 7664-7668
• 12 Sorrentino R, Iannicola C, Costanzi S, Chersi A, Tosi R. Detection of complex alleles by direct analysis of DNA heteroduplexes. Immunogenetics 1991; 33: 118-123
• 13 White MB, Carvalho M, Derse D, O'Brien SJ, Dean M. Detecting single base substitutions as heteroduplex polymorphisms. Genomics 1992; 12: 301-306