TY - JOUR
T1 - 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
AU - Higuchi, Miyoko
AU - Kazazian, Haig H.
AU - Kasch, Laura
AU - Warren, Tina C.
AU - McGinniss, Matthew J.
AU - Phillips, John A.
AU - Kasper, Carol
AU - Janco, Robert
AU - Antonarakis, Stylianos E.
PY - 1991
Y1 - 1991
N2 - Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.
AB - Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.
KW - Denaturing gradient gel electrophoresis
KW - Polymerase chain reaction
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U2 - 10.1073/pnas.88.16.7405
DO - 10.1073/pnas.88.16.7405
M3 - Article
C2 - 1908096
AN - SCOPUS:0025820639
SN - 0027-8424
VL - 88
SP - 7405
EP - 7409
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 16
ER -