Mutation screening of the F VIII gene in 10 hemophilia A families

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis. Key words: hemophilia A;  coagulation factor FⅧ;  gene diagnosis;  linkage analysis